Transgenic plants that exhibit enhanced nitrogen assimilation

ABSTRACT

The present invention relates to a method for producing plants with improved agronomic and nutritional traits. Such traits include enhanced nitrogen assimilatory and utilization capacities, faster and more vigorous growth, greater vegetative and reproductive yields, and enriched or altered nitrogen content in vegetative and reproductive parts. More particularly, the invention relates to the engineering of plants modified to have altered expression of key enzymes in the nitrogen assimilation and utilization pathways. In one embodiment of the present invention, the desired altered expression is accomplished by engineering the plant for ectopic overexpression of one of more the native or modified nitrogen assimilatory enzymes. The invention also has a number of other embodiments, all of which are disclosed herein.

This invention was made with government support under grant no.: GM32877awarded by the National Institute of Health, and grant nos.: DEFG0292and ER20071 awarded by the Department of Energy. The government hascertain rights in the invention.

This application is a continuation of application Ser. No. 08/319,176filed Oct. 6, 1994, now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/132,334 filed Oct. 6, 1993, now abandoned, thedisclosures of both of which are hereby incorporated by reference intheir entirety.

1. INTRODUCTION

The present invention relates generally to genetic-engineering plants todisplay enhanced nitrogen assimilatory and utilization capacities, growlarger, more efficiently or rapidly, and/or have enriched nitrogencontents in vegetative and/or reproductive plant parts and/or increasedbiomass. More particularly, this invention relates to producingtransgenic plants engineered to have altered expression of key enzymesin the nitrogen assimilation and utilization pathways. The engineeredplants may be productively cultivated under conditions of low nitrogenfertilizer input or in nitrogen poor soils. Alternatively, theengineered plants may be used to achieve faster growing or maturingcrops, higher crop yields and/or more nutritious products under idealcultivation conditions.

2. BACKGROUND OF THE INVENTION

Nitrogen is often the rate-limiting element in plant growth and allfield crops have a fundamental dependence on inorganic nitrogenousfertilizer. Since fertilizer is rapidly depleted from most soil types,it must be supplied to growing crops two or three times during thegrowing season. Nitrogenous fertilizer, which is usually supplied asammonium nitrate, potassium nitrate, or urea, typically accounts for 40%of the costs associated with crops such as corn and wheat. It has beenestimated that approximately 11 million tons of nitrogenous fertilizeris used in both North America and Western Europe annually, costingfarmers $2.2 billion each year (Sheldrick, 1987, World Nitrogen Survey,Technical Paper no. 59, Washington, D.C.). Furthermore, World Bankprojections suggest that annual nitrogen fertilizer demand worldwidewill increase from around 90 million tons to well over 130 million tonsover the next ten years. Increased use efficiency of nitrogen by plantsshould enable crops to be cultivated with lower fertilizer input, oralternatively on soils of poorer quality and would therefore havesignificant economic impact in both developed and developingagricultural systems.

Using conventional selection techniques plant breeders have attempted toimprove nitrogen use efficiency by exploiting the variation available innatural populations of corn, wheat, rice and other crop species. Thereare, however, considerable difficulties associated with the screening ofextensive populations in conventional breeding programs for traits whichare difficult to assess under field conditions, and such selectionstrategies have been largely unsuccessful.

2.1. Nitrogen Assimilatory Pathway in Plants

Plants obtain nitrogen from their environment in the form of inorganiccompounds, namely nitrate and ammonia taken up from roots, andatmospheric N₂ reduced to ammonia in nitrogen-fixing root nodules.Although some nitrate and ammonia can be detected in the transportingvessels (xylem and phloem), the majority of nitrogen is firstassimilated into organic form (e.g., amino acids) which are thentransported within the plant.

The first step in the assimilation of inorganic nitrogen into organicform predominately involves the incorporation of ammonia with glutamateto form lutamine, catalyzed by the enzyme, glutamine synthetase (GS; EC6.3.1.2). Glutamine thus formed may in turn donate its amide group inthe formation of asparagine, catalyzed by the enzyme, asparaginesynthetase (AS; E.C. 6.3.5.4). The steady flow of nitrogen from ammoniato asparagine in this pathway depends upon the recycling of glutamateand α-ketoglutarate and aspartate, catalyzed by glutamine 2:oxoglutarateaminotransferase (GOGAT; E.C.) and aspartate aminotransferase (AspAT;E.C.), respectively (see FIG. 1). Thus, GS, AS, AspAT and GOGAT comprisethe key enzymes of the main nitrogen assimilatory pathway of higherplants.

Evidence exists indicating that ammonia incorporation may proceedthrough alternative pathways other than that catalyzed by GS (FIG. 1).See Knight and Langston-Unkefer, 1988, Science 241:951-954. One pathwaymay involve the incorporation of ammonia with α-ketoglutarate to formglutamate, catalyzed by glutamate dehydrogenase (GDH). Another pathwaymay involve the incorporation of ammonia with aspartate to formasparagine, catalyzed by asparagine synthetase (Oaks and Ross, 1984,Can. J. Bot. 62:68-73; Stulen and Oaks, 1977, Plant Physiol.60:680-683). Since both of these enzymes (GDH and AS) have a high Km forammonia, the roles of these alternative nitrogen assimilation pathwaysunder normal growth conditions (e.g., low concentrations of internalammonia) remain unclear. One study suggests these or other alternativenitrogen assimilation pathways may make significant contributions to aplant's nitrogen assimilation capacity when intracellular ammoniumconcentration is elevated above normal levels (Knight andLangston-Unkefer, id.).

2.2. Nitrogen Transport and Utilization

Glutamine and asparagine represent the major long-distance nitrogentransport compounds in plants and are abundant in phloem sap. Aside fromtheir common roles as nitrogen carriers, these two amino acids havesomewhat different roles in plant nitrogen metabolism. Glutamine is themore metabolically active of the two and can directly donate its amidenitrogen to a large number of substrates in various anabolic reactions.Because of its reactivity, glutamine is generally not used by plants tostore nitrogen.

By contrast, asparagine is a more efficient compound for nitrogentransport and storage compared to glutamine because of its higher N:Cratio. Furthermore, asparagine is also more stable than glutamine andcan accumulate to higher levels in vacuoles. Indeed, in plants that havehigh nitrogen assimilatory capacities, asparagine appears to play adominant role in the transport and metabolism of nitrogen. See Lea andMiflin, Transport and metabolism of asparagine and other nitrogencompounds within the plant, in The Biochemistry of Plants: AComprehensive Treatise, vol 5. Amino acid and derivatives, Miflin ed.,Academic Press, New York (1980) pp 569-607; and Sieciechowicz et al.,1988, Phytochemistry 27:663-671. Because of its relative stability,asparagine does not directly participate in nitrogen metabolism, butmust be first hydrolyzed by the enzyme asparaginase (ANS; E.C. 3.5.1.1)to produce aspartate and ammonia which then could be utilized insynthesis of amino acids and proteins (See FIG. 1).

2.3. Plant Genes Involved in Nitrogen Assimilation and Utilization

Many of the genes encoding enzymes involved in plant nitrogenassimilation and utilization have been cloned and studied. See Tsai andCoruzzi, Transgenic Plants for Studying Genes Encoding Amino AcidBiosynthetic Enzymes, in Transgenic Plants, Vol. 1, Kung and Wu eds.,Academic Press, San Diego, Calif., (1993) pg 181-194, and referencescited therein for discussions of plant glutamine synthetase (GS) andasparagine synthetase (AS) genes; Udvardi and Kahn, 1991, Mol. Gen.Genet. 231:97-105, for a discussion of the alfalfa aspartateaminotransferase gene; Zehnacker et al., 1992, Planta 187:266-274, for adiscussion of the tobacco glutamate 2:oxoglutarate aminotransferase(GOGAT, also known as glutamate synthetase) gene; Lough et al, 1992,Plant Mol. Biol. 19:391-399, and Dickson et al., 1992, Plant Mol. Biol.20:333-336, for discussions of lupin asparaginase gene.

Among the plant nitrogen assimilation and utilization genes, the mostextensively studied are the glutamine synthetase and asparaginesynthetase genes. Multiple genes exist for GS and AS, and molecularcharacterization of these genes has shown that they have differentexpression patterns.

2.3.1. Glutamine Synthetase Genes

GS is active in a number of organs during plant development (McNally etal., 1983, Plant Physiol. 72:22-25). In roots it assimilates ammoniaderived from soil water (Oaks and Hirel, 1985, Ann. Rev. Plant Physiol.36:345-365), and in root nodules of legumes, GS assimilates ammoniafixed by rhizobia (Cullimore et al. 1983, Planta 157:245-253). Incotyledons GS reassimilates nitrogenous reserves mobilized duringgermination (Lea and Joy, 1983, Amino acid interconversion ingerminating seeds. In: Recent Advances in Phythochemistry: Mobilizationof Reserves in Germination, ed. Nozolillo et al., Plenum Press, p.77-109), and in leaves chloroplastic GS2 assimilates ammonia released inphotorespiration (Givan et al. 1988, TIBS 13:433-437). The various rolesof GS are undertaken by different GS isoforms which are derived fromdifferent genes that are expressed differentially (Gebhardt et al. 1986,EMBO J. 5:1429-1435; Tingey et al. 1987, EMBO J. 6:1-9).

In pea, Phaseolus, and Arabidopsis, chloroplastic GS2 is encoded by asingle nuclear gene, whereas multiple genes for cytosolic GS exist ineach of these species (Bennett et al. 1989, Plant Mol. Biol. 12:553-565;Tingey et al. 1988, J. Biol. Chem. 263:9651-9657; Peterman and Goodman,1991, Mol. Gen. Genet. 230:145-154). The analysis of the expression ofthese GS genes in vivo and in transgenic host plants has helped unravelthe roles of the various GS isoforms in plant nitrogen metabolism.

The GS gene family in pea comprises four distinct but homologous nucleargenes. Three encode cytosolic GS isoforms, and one encodes thechloroplastic GS2 isoform (Tingey et al., 1987, EMBO J. 6:1-9; Tingey etal., 1988, J. Biol. Chem. 263:9651-9657). Northern blot analysis hasdemonstrated that the gene for chloroplastic GS2 is expressed in leavesin a light-dependent fashion due in part to phytochrome and in part tophotorespiratory effects (Edwards and Coruzzi, 1989, Plant Cell1:241-248). The three genes for cytosolic GS (GS1, GS3A and GS3B) alsoappear to serve distinct roles. In roots cytosolic GS1 is thepredominant isoform, although it is also expressed in nodules. CytosolicGS3A and GS3B are highly expressed in nodules and also in cotyledons ofgerminating seeds (Tingey et al., 1987, EMBO J. 6:1-9; Walker andCoruzzi, 1989, Plant Physiol. 91:702-708). While the GS3A and GS3B genesare near identical in sequence, gene specific S1-nuclease analysis hasrevealed that GS3A expression is consistently higher than that of GS3B(Walker and Coruzzi, 1989, Plant Physiol. 91:702-708). Usingpromoter-GUS fusions and transgenic plant analysis it has been shownthat chloroplastic GS2 is expressed only in photosynthetic cell-typesand that cytosolic GS3A is expressed exclusively in the phloem cells ofthe vasculature in most organs. GS3A is also strongly expressed in rootand nodule meristems (Edwards et al., 1990, Proc. Natl. Acad. Sci. USA.87:3459-3463; Brears et al., 1991, The Plant Journal, vol. 1, pp.235-244). From the tightly controlled regulation at cell-type and organlevel it appears that the various genes for GS fulfill non-overlappingroles in ammonia assimilation.

2.3.2. Asparagine Synthetase Genes

Two AS genes have been cloned from pea (AS1 and AS2); both are expressedat highest levels in root nodules and cotyledons. AS1 and AS2 are bothexpressed in roots. AS2 is expressed constitutively in roots, while AS1is expressed only in roots of dark-grown plants (Tsai and Coruzzi, 1990,EMBO J 9:323-332). Furthermore, AS1 and AS2 are expressed in matureleaves of dark-adapted plants, whereas their expression is inhibited bylight. This high level of AS gene expression in the dark corresponds tothe use of asparagine as a long-distance nitrogen transport compoundsynthesized under conditions of reduced availability of photosyntheticcarbon (asparagine has a higher N:C ratio than glutamine). Studies ofAS1 promoter-GUS fusions in transgenic plants have shown that the AS1gene, like the GS3A gene, is also expressed exclusively in phloem cells.From the tightly controlled regulation at cell-type and organ level, itseems that the various AS genes may also fulfill non-overlapping rolesin plant nitrogen metabolism.

2.4. Genetic Engineering of Nitrogen Assimilation and UtilizationProcesses in Plants

In plants, genetic engineering of nitrogen assimilation processes hasyielded varied results. In one case, expressing a prokaryotic ammoniumdependent asparagine synthetase (ASN-A) gene in tobacco conferredresistance to various glutamine synthetase (GS) inhibitors (Dudits etal., Transgenic plants expressing a prokaryotic ammonium dependentasparagine synthetase, WO 9111524, Aug. 8, 1991). These same plants alsoexhibited a number of growth alterations including increased growthrate, accelerated plant development, early flower development andincreased green mass and plant dry weight. The growth effect of ASN-Aexpression is paradoxical as GS inhibitor treatments enhanced ratherthan attenuated growth in the engineered plants.

By contrast, numerous studies examining overexpression of glutaminesynthetase (GS) have failed to report any positive effect of theoverexpression on plant growth. See Lea and Forde, 1994, Plant Molec.Biol. 17:541-558; Eckes et al., 1989, Molec. Gen. Genet. 217:263-268(transgenic tobacco plants overexpressing alfalfa GS); Hemon et al.,1990, Plant Mol. Biol. 15:895-904 (transgenic tobacco plantsoverexpressing bean GS in the cytoplasm or mitochondria); Hirel et al.,1992, Plant Mol. Biol. 20:207-218 (transgenic tobacco plantsoverexpressing soybean GS in tobacco plants). One study has reportedobserving increases in total soluble protein content in transgenictobacco plants overexpressing the alfalfa GS1 gene. However, since thissame study also reported similar increases in total soluble proteincontent in transgenic tobacco plants expressing antisense RNA to the GS1gene, the relationship between GS1 expression and the increase insoluble protein appears unclear (Temple et al., 1993, Mol. Gen. Genet.236:315-325). One clearly established effect of GS overexpression inplants is resistance to phosphinothricin, a GS inhibiting herbicide(Eckes et al. ibid.; Donn et al., 1984, J. Molec. Appl. Genet. 2:621-635(a phosphinothricin-resistant alfalfa cell line contained amplificationof the GS gene)). There also has been a claim that plants engineeredwith overexpression of an alfalfa GS gene grow more rapidly thanunengineered plants (Eckes et al., 1988, Australian Patent OfficeDocument No.: AU-A-17321/88). The claimed faster growth, however, occursonly under low- but not normal- or high-nitrogen growth conditions.Moreover, it is unclear whether the faster growth produce mature plantswith greater biomass or reproductive yield. Compare id. with Eckes etal., 1989, Molec. Gen. Genet. 217:263-268.

3. SUMMARY OF THE INVENTION

The present invention relates to the production of transgenic plantswith altered expression levels and/or cell-specific patterns ofexpression of key enzymes involved in nitrogen assimilation andutilization (The respective roles of these enzymes are shown in FIG. 1)so that the resulting plants have enhanced nitrogen assimilation and/orutilization capacities as well as improved agronomic characteristics.The present invention particularly relates to altering the expression ofglutamine synthetases, asparagine synthetases, glutamate 2:oxoglutarateaminotransferases (glutamate 2:oxoglutarate aminotransferase is alsoknown as glutamate synthetase), aspartate aminotransferases, glutamatedehydrogenases and asparaginases (see FIG. 1).

The invention has utility in improving important agronomiccharacteristics of crop plants. One of the improvements would be theability of the engineered plants to be productively cultivated withlower nitrogen fertilizer inputs and on nitrogen-poor soil. Additionalimprovements include more vigorous (i.e., faster) growth as well asgreater vegetative and/or reproductive yield under normal cultivationconditions (i.e., non-limiting nutrient conditions). To achieve thesesame improvements, traditional crop breeding methods would requirescreening large segregating populations. The present inventioncircumvent the need for such large scale screening by producing plantsmany of which, if not most, would have the desired characteristics.

According to the present invention, achieving the desired plantimprovements may require, in some instances, the ectopic overexpressionof a single gene or multiple genes encoding nitrogen assimilation orutilization enzyme(s). The modified expression may involve engineeringthe plant with any or several of the following: a) a transgene in whichthe coding sequence for the enzyme is operably associated to a strong,constitutive promoter; b) additional copies of the native gene encodingthe desired enzyme; c) regulatory gene(s) that activates the expressionof the desired gene(s) for nitrogen assimilation or utilization; d) acopy of the native gene that has its regulatory region modified forenhanced expression; and e) a transgene which expresses a mutated,altered or chimeric version of a nitrogen assimilation or utilizationenzyme.

In other instances, achieving the desired plant improvements may requirealtering the expression pattern of a nitrogen assimilation orutilization enzyme. The altered expression pattern may involveengineering the plant with any or many of the following: a) a transgenein which the coding sequence for the enzyme is operably associated to apromoter with the desired expression pattern (such promoters may includethose considered to have tissue or developmental-specific expressionpatterns); b) modified regulatory genes that activates the expression ofthe enzyme-encoding gene in the preferred pattern; c) a native copy ofthe enzyme-encoding gene that has its regulatory region modified toexpress in the preferred pattern.

In yet other instances, achieving the desired plant improvements mayrequire suppressing the expression level and/or pattern of a nitrogenassimilation or utilization enzyme. The suppression of expression mayinvolve engineering the plant with genes encoding antisense RNAs,ribozymes, co-suppression constructs, or "dominant negative" mutations(see Herskowitz, 1987, Nature 329:219-222 for an explanation of themechanism of gene suppression by dominant negative mutations). Further,gene suppression may also be achieved by engineering the plant with ahomologous recombination construct that replaces the native gene with acopy of a defective gene or enzyme-encoding sequence that is under thecontrol of a promoter with the desired expression level and/or pattern.

In still other instances, achieving the desired plant improvements mayrequire expressing altered or different forms of the enzymes in thenitrogen assimilation or utilization pathways. Such efforts may involvedeveloping a plant-expressible gene encoding a nitrogen assimilation orutilization enzyme with catalytic properties different from those of thecorresponding host plant enzymes and engineering plants with that geneconstruct. Gene sequences encoding such enzymes may be obtained from avariety of sources, including, but not limited to bacteria, yeast,algae, animals, and plants. In some cases, such coding sequences may bedirectly used in the construction of plant-expressible gene fusions byoperably linking the sequence with a desired plant-active promoter. Inother cases, the utilization of such coding sequences in gene fusionsmay require prior modification by in vitro mutagenesis or de novosynthesis to enhance their translatability in the host plant or to alterthe catalytic properties of the enzymes encoded thereon. Usefulalterations may include, but are not limited to, modifications ofresidues involved in substrate binding and/or catalysis. Desiredalterations may also include the construction of hybrid enzymes. Forinstance, the different domains of related enzymes from the sameorganism or different organisms may be recombined to form enzymes withnovel properties.

In all instances, a plant with the desired improvement can be isolatedby screening the engineered plants for altered expression pattern orlevel of the nitrogen assimilation or utilization enzyme, alteredexpression pattern or level of the corresponding mRNA or protein,altered nitrogen assimilation or utilization capacities, increasedgrowth rate, enhanced vegetative yield, or improved reproductive yields(e.g., more or larger seeds or fruits). The screening of the engineeredplants may involve enzymatic assays and immunoassays to measureenzyme/protein levels; Northern analysis, RNase protection, primerextension, reverse transcriptase/PCR, etc. to measure mRNA levels;measuring the amino acid composition, free amino acid pool or totalnitrogen content of various plant tissues; measuring growth rates interms of fresh weight gains over time; or measuring plant yield in termsof total dry weight and/or total seed weight.

The present invention is based, in part, on the surprising finding thatenhancing the expression of nitrogen assimilation or utilization enzymesin plants resulted in enhanced growth characteristics, or improvedvegetative or reproductive yields. The invention is illustrated hereinby the way of working examples in which tobacco plants were engineeredwith recombinant constructs encoding a strong, constitutive plantpromoter, the cauliflower mosaic virus (CaMV) 35S promoter, operablylinked with sequences encoding a pea glutamine synthetase (GS) gene or apea asparagine synthetase (AS) gene. RNA and protein analyses showedthat a majority of the engineered plants exhibited ectopic,overexpression of GS or AS. The GS or AS overexpressing lines havehigher nitrogen contents, more vigorous growth characteristics,increased vegetative yields or better seed yields and quality than thecontrol, wild-type plant.

3.1. Definitions

The terms listed below, as used herein, will have the meaning indicated.

    ______________________________________                                        35S         =      cauliflower mosaic virus promoter for                                         the 35S transcript                                         AS          =      Asparagine synthetase                                      AspAT       =      aspartate aminotransferase (also                                              known as AAT)                                              CaMV        =      Cauliflower Mosaic Virus                                   cDNA        =      complementary DNA                                          DNA         =      deoxyribonucleic acid                                      GDH         =      glutamate dehydrogenase                                    gene fusion =      a gene construct comprising a                                                 promoter operably linked to a                                                 heterologous gene, wherein said                                               promoter controls the transcription                                           of the heterologous gene                                   GOGAT       =      glutamate 2:oxoglutarate                                                      aminotransferase (alternately known                                           as glutamate synthetase)                                   Fd-GOGAT    =      Ferredoxin-dependent glutamate                                                synthase                                                   NADH-GOGAT  =      NADH-dependent glutamate synthase                          GS          =      glutamine synthetase                                       heterologous                                                                              =      In the context of gene constructs, a                       gene               heterologous gene means that the gene                                         is linked to a promoter that said                                             gene is not naturally linked to. The                                          heterologous gene may or may not be                                           from the organism contributing said                                           promoter. The heterologous gene may                                           encode messenger RNA (mRNA),                                                  antisense RNA or ribozymes.                                nitrogen    =      A nitrogen non-limiting growth                             non-limiting       condition is one where the soil or                         growth condition   medium contains or receives                                                   sufficient amounts of nitrogen                                                nutrients to sustain healthy plant                                            growth. Examples of nitrogen non-                                             limiting growth conditions are                                                provided in section 5.2.3. Moreover,                                          one skilled in the art would                                                  recognize what constitutes such                                               soils, media and fertilizer inputs                                            for most species and varieties of                                             important crop and ornamental plants                                          (see section 5.3.).                                        PCR         =      polymerase chain reaction                                  Progenitor  =      untransformed, wild-type plant                             plant                                                                         RNA         =      ribonucleic acid                                           ______________________________________                                    

4. DESCRIPTION OF THE FIGURES

FIG. 1. Pathway of nitrogen assimilation/metabolism in plants. The majorroute for nitrogen assimilation is via glutamine synthetase (GS) andglutamate synthase (GOGAT). Glutamate dehydrogenase (GDH) is thought tofunction under conditions of ammonia toxicity in the biosynthetic role,or may provide catalytic amounts of glutamate to fuel the GS/GOGATcycle. GDH probably is more active in its catalytic role to releaseammonia from glutamate (e.g., during germination). Aspartate aminotransferase (AspAT) catalyzes a reversible reaction. Asparaginesynthetase (AS) has two activities; a glutamine-dependent activity andan ammonia-dependent activity. Asparagine catabolism occurs viaasparaginase (ANS) to liberate aspartate and ammonia.

FIG. 2. Engineering a chimeric Fd/NADH GOGAT enzyme. Plantferredoxin-GOGAT (Fd-GOGAT) large subunit contains Fd-Binding domain(diagonal cross-bars). Plant and E. coli NADH-GOGAT: large subunit (openbar), small subunit contains NADH-binding domain (vertical hatches).Chimeric Fd/NADH GOGAT is engineered to contain the large subunit ofFd-GOGAT (Fd-binding domain) plus the small subunit of the NADH-GOGAT ofeither plant or E. coli. The engineering is done by making an in-frametranslational fusion of a sequence encoding a plant Fd-GOGAT and asequence encoding a small subunit of a plant or E. coli NADH-GOGAT,containing the NADH-binding domain. The chimeric protein encodes abispecific or bifunctional GOGAT enzyme which can utilize either Fd orNADH as the reductant.

FIG. 3. Maps of Binary Plant Expression Vectors. The binary expressionvectors pTEV4, pTEV5, pTEV8 and pTEV9 are derivatives of pBIN19 (Bevan,1984, Nucleic Acids Res. 12:8711-8721) constructed for the high levelexpression of cDNAs in transgenic tobacco. For details of constructionsee Section 6.1.1.

FIG. 4. Chimeric 35S CaMV-GS cDNA Constructs Transferred to TransgenicTobacco. Pea GS cDNAs were cloned into pTEV expression vectors (see FIG.3, and Section 6.1.1) for expression behind the Strasbourg strain CaMV35S promoter (35S). For GS3A and GS2, "modified" clones were constructedincorporating introns from the genomic sequence into the cDNAs (seeSection 6.1.2.). Sources of the GS cDNA clones were: GS2 (also known as(aka) GS185); GS1 (aka GS299); GS3A (aka GS341) (Tingey et al., 1988, J.Biol. Chem. 263:9651-9657; Tingey et al., 1987, EMBO J. 6:1-9).

FIG. 5. Analysis of GS Protein in Primary (T1) Transformants ContainingGS Transgenes. Top panel: Western analysis of GS polypeptides in primarytransformants. Lanes 1 and 2: primary transformants Z17-6 and Z17-12carrying the cytosolic GS3A gene show overexpression and co-suppressionphenotypes respectively. Lanes 3-6: primary transformants Z41-20, Z54-2,Z54-7, and Z54-8 carrying the chloroplastic GS2 gene are allco-suppressed for chloroplast GS2 (cf. GS). Controls are: TL--tobaccoleaf, PL--pea leaf, and PR--pea root. Total GS activities are shown (aspercentages relative to controls =(100%)) below the Western panel.Bottom panel: Coomassie staining of RUBISCO large subunit proteindemonstrating approximately equal loading of samples. ctGS-chloroplasticGS2 (˜45 kD); cyGS-cytosolic GS (˜38 kD).

FIGS. 6A-C. Analysis of GS Protein, RNA and Holoenzyme from T2 ProgenyTransgenic Plants Containing Pea GS Transgenes. Of the four T2 plantsfrom each primary transformant typically analyzed, a singlerepresentative plant was included in this figure. In the case of Z17-9,the T2 progenies showed two different profiles and both are shown(Z17-9A and Z17-9B). Controls: TL/T--tobacco leaf, P--pea leaf. Panel A(upper): Western analysis of GS polypeptides in transgenic plants. PanelA (lower): Coomassie staining of RUBISCO large subunit protein to showapproximately equal loading of samples. Panel B (upper): Northern blotshybridized with the approximate cDNA probes for GS1 (left), GS3A(center), and GS2 (right). Panel B (lower): Control hybridization withthe pea rRNA gene probe. Panel C: Non-denaturing gel and GS activityanalysis showing GS holoenzymes A*, B, and C in transgenic plants. GSactivities are expressed as percentages compared to controls(control=100% activity).

FIG. 7A. Activity Gel Analysis of GS Holoenzymes. Protein extracts frompea chloroplast (PC), pea root (PR), tobacco chloroplast (TC) andtobacco roots (TR) demonstrating the migration of chloroplastic- andcytosolic-enriched GS protein samples relative to the migration of theholoenzymes of GS1 and GS3A overexpressing plants. Lane 1: peachloroplast protein (PC) has GS holoenzyme B only; lane 2: pea rootprotein (PR) has GS holoenzyme C only; lane 3: tobacco chloroplastprotein (TC) has GS holoenzyme B only; lane 4: tobacco root protein hasGS holoenzyme C only. Lane 5: protein from plant Z17-7 (carrying the35S-GS3A construction) has GS holoenzymes A* and B; lane 5: protein fromplant Z3-1 (carrying the 35S-GS1 construction) has GS holoenzymes B andC.

FIG. 7B. Western Analysis of GS Proteins Isolated from GS HoloenzymesA*, B, and C. Holoenzymes A* and C observed in transgenic tobaccooverexpressing GS3A and GS1 were excised from non-denaturing gels,re-extracted in protein isolation buffer, and electrophoresed underdenaturing conditions for Western analysis using GS antibodies. Lane 1:tobacco leaf protein as control; lane 2: GS holoenzyme A* from Z17-7;lane 3: isolated chloroplast GS2 (holoenzyme B) as control; lane 4: GSholoenzyme C from Z3-1.

FIG. 8. Western and Northern Analysis of GS Protein and RNA inTransgenic Plants Selected for Growth Analysis Ectopically Expressingeither Cytosolic GS1 or GS3A. Upper panel: Western blot for GS proteins.Lower panel: Northern blot for GS mRNA. Pl and Tl are pea and tobaccoleaf controls. Lanes 1 and 2, and 5 and 6 are plants overexpressing GS1,and lanes 3 and 4, and 7 and 8 are plants overexpressing GS3A.Transgenic plants to the left of the broken line were analyzed in growthexperiment A, and those to the right were analyzed in growth experimentB. Corresponding probes were used in the Northern blot; the left peacontrol was hybridized to GS1, and the right-hand pea control washybridized to GS3A.

FIGS. 9A-B. Increase in fresh weight of transgenic lines overexpressingcytosolic GS1 (Z3) or cytosolic GS3A (Z17). Panel A: The results ofexperiment A with transgenic lines Z3-1, Z3-2, Z17-6, Z17-7, and anon-transformed control (C). Panel B: The results of experiment B withtransgenic lines Z3-3, Z3-4, Z17-3, Z17-11, and two non-transformedcontrols (C1 and C2). This is a graphic representation of data shown inTable 2, and analyzed statistically in Table 3.

FIG. 10. Qualitative growth pattern of plants with altered GS expressionpatterns. Plants in each panel were sown at the same time and grown insoil for approximately three weeks. Control panel: SR1 untransformedtobacco (100% GS activity). Z3-A1 panel: Transgenic plants withoverexpress GS1 (123% GS activity). Z17-B7 panel: Transgenic plant whichoverexpresses GS3 (107% GS activity). Z54-A2 Panel: Transgenic plantco-suppressed for GS2 (28% GS activity).

FIGS. 11A and 11B. Linear relationship between GS activity and plantfresh weight or total leaf protein. T2 progenies of primarytransformants which showed no segregation of the Kan^(R) phenotypeassociated with the transgene were selected for growth analysis. Kan^(R)T2 plants were selected on MSK media (R. B. Horsch, et al., Science227:1229 (1985)) and transferred to sand at 18 days. Plants weresubirrigated and surface fed every two days with 50 mls of 1X Hoagland'ssolution (D. R. Hoagland et al., Circ. Calif. Agric. Exp. Stn. 347:461(1938)) containing 10 mM KNO₃. For each line, eight T2 progenies wereanalyzed individually for total plant fresh weight (grams), specificactivity of total leaf GS as determined by the transferase assay (B. M.Shapiro, et al., Methods Enzymol. 17A:910 (1970)) and protein/gram freshweight. Plants analyzed were: Control, SR1 untransformed tobacco; Z54-4co-suppressed by GS2; Z17-7 overexpressing GS3A; Z3-1 overexpressingGS1. FIG. 11A; Plant fresh weight vs. GS activity. FIG. 11B; protein/gmfresh weight vs. GS activity.

FIG. 12. Chimeric 35 S CaMV-AS Constructs Transferred to Transgenictobacco. cDNAs for the AS1 gene and the glnΔAS1 gene were fused to the35S promoter and nopaline synthase transcriptional terminator fortransfer to tobacco using the binary expression vector pTEV5.

FIG. 13. Northern analysis of transgenic plants expressing either AS1 orglnΔAS1. 10 μg of total RNA isolated from leaves of individualtransformants was loaded in leach lane. Blots were probed with the AS1cDNA from pea. A positive control includes AS mRNA in dark-grown pealeaves (PL). A negative control includes AS mRNA in light-grown tobaccoleaves (TL).

FIG. 14. Increase in fresh weight of transgenic lines overexpressing AS1and glnΔAS1 is expressed graphically from week 3 to week 6post-germination. This is a graphic representation of data shown inTable (5) and analyzed statistically in Table (6).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetic engineering of nitrogenmetabolism in plants. In particular, the invention relates to alteringthe enzymes involved in nitrogen assimilation or utilization and/ortheir expression in order to engineer plants with better growthcharacteristics, enriched nutritional qualities, improved vegetative andyield and/or enhanced seed yield or quality.

Accordingly--without intending to be limited to a particularmechanism--the targets for engineering are genes encoding for enzymesinvolved in the assimilation of ammonia into the amino acids, glutamine,aspartate, asparagine or glutamate, or in the utilization of these sameamino acids in biosynthetic reactions. The target genes include thoseencoding glutamine synthetase (GS), asparagine synthetase (AS),glutamate 2:oxoglutarate aminotransferase (GOGAT), aspartateaminotransferase (AspAT), glutamate dehydrogenase (GDH) and asparaginase(ANS). See FIG. 1 for a diagram of the roles played by of these enzymesin nitrogen assimilation and utilization.

These enzymes can be altered or their expression can be enhanced,suppressed or otherwise modified (e.g., ectopic expression) to engineera plant with desirable properties. The engineering is accomplished bytransforming plants with nucleic acid constructs described herein. Thetransformed plants or their progenies are screened for plants thatexpress the desired altered enzyme or exhibit the desired alteredexpression of the nitrogen assimilation or utilization enzyme, alteredexpression of the corresponding mRNA, altered nitrogen assimilation orutilization capacities, increased growth rate, enhanced vegetativeyield, and/or improved reproductive yields.

Engineered plants exhibiting the desired physiological and/or agronomicchanges can be used in plant breeding or directly in agriculturalproduction. These plants having one altered enzyme also may be crossedwith other altered plants engineered with alterations in the othernitrogen assimilation or utilization enzymes (e.g., cross a GSoverexpressing plant to an AS overexpressing plant) to produce lineswith even further enhanced physiological and/or agronomic propertiescompared to the parents.

The invention is illustrated by working examples of plants engineeredfor ectopic, overexpression of GS or AS. In all instances, engineeredplants that exhibit ectopic, overexpression of GS or AS also show bettergrowth characteristics, enriched nutritional qualities, improvedvegetative yield and/or enhanced seed quality or yield over control,wild-type plants.

5.1. Alteration of Nitrogen Assimilatory and Utilization Pathways

In accordance with one aspect of the present invention, desirable plantsmay be obtained by engineering ectopic overexpression of enzymesinvolved in initial assimilation of ammonia into amino acids glutamine,asparagine or glutamate and further conversion to aspartate. The termectopic is used herein to mean abnormal subcellular (e.g., switchbetween organellar and cytosolic localization), cell-type, tissue-typeand/or developmental or temporal expression (e.g., light/dark) patternsfor the particular gene or enzyme in question. Such ectopic expressiondoes not necessarily exclude expression in tissues or developmentalstages normal for said enzyme but rather entails expression in tissuesor developmental stages not normal for the said enzyme. The termoverexpression is used herein to mean above the normal expression levelin the particular tissue, all and/or developmental or temporal stage forsaid enzyme.

Key enzymes involved in assimilation of ammonia into glutamine and itsfurther metabolism into glutamate, aspartate, and asparagine are:glutamine synthetase, asparagine synthetase, glutamate 2:oxoglutarateaminotransferase, aspartate aminotransferase, glutamate dehydrogenaseand asparaginase. The present invention provides that engineeringectopic overexpression of one or more of these enzymes would produceplants with the desired physiological and agronomic properties. In apreferred embodiment, a plant is engineered for the ectopicoverexpression of glutamine synthetase or asparagine synthetase. For GS,where cytosolic and chloroplastic forms of an enzyme exist, engineeringof enhanced expression of the cytosolic form is preferred. The cytosolicform of GS includes both nodule-specific (e.g., pea GS3A & B) androot-specific (e.g., pea GS1) enzymes. The engineering of enhancedexpression of "root-specific" cytosolic GS (e.g., pea GS1) is especiallypreferred. The present invention also provides for engineering thatalters the subcellular localization of said enzyme. For example,engineering a chloroplast target sequence onto a cytosolic enzyme suchas AS, may improve nitrogen assimilation in plants. This would beespecially valuable in mesophyll cells to reassimilate photorespiratoryammonia.

In accordance to another aspect of the present invention, desirableplants may be obtained by engineering enhanced ammonia incorporationthough an alternate nitrogen assimilation pathway. In particular, theengineering is accomplished by suppressing the normal, major route ofnitrogen assimilation through glutamine synthetase. In plant speciesthat encode multiple GS isozymes, this may require the suppression ofthe endogenous GS genes. In preferred embodiments, a plant engineeredwith suppressed GS expression is further engineered for ectopicoverexpression of an alternative N-assimilatory enzyme such asasparagine synthetase (AS) and/or glutamine dehydrogenase (GDH). In mostpreferred embodiments, the GS and AS/GDH engineered plant isadditionally engineered for enhanced expression of one or more of theother enzymes involved in nitrogen assimilation or utilization processes(see FIG. 1).

In accordance with a third aspect of the present invention, desirableplants may be obtained by engineering ectopic overexpression of anenzyme involved in the utilization of assimilated nitrogen. Embodimentsof this aspect of the present invention may involve engineering plantswith ectopic overexpression of enzymes catalyzing the use of glutamine,glutamate and asparagine in catabolic reactions. In a preferredembodiment, a plant is engineered for the ectopic overexpression ofasparaginase.

In accordance with a fourth aspect of the present invention, desirableplants may be obtained by engineering the expression of an altered,mutated, chimeric, or heterologous form of an enzyme involved in theassimilation or utilization of nitrogen. Embodiments of this aspect ofthe present invention may involve engineering plants to express nitrogenassimilation or utilization enzymes from a heterologous source (ie. anenzyme from a different plant or organism, including animals andmicrobes). Additional embodiments may involve developing nitrogenassimilation or utilization enzymes that have increased efficiencies,for example, in substrate binding, catalysis, and/or product release andengineering plants to express such novel enzymes. These novel enzymesmay be developed by in vitro mutagenesis of key amino acid residuesaffecting the aforementioned processes. Alternatively such novel enzymesmay be developed by recombining domains from related enzymes. Forexample, a chimeric bifunctional GOGAT enzyme could be engineered tocontain both ferredoxin- and NADH-GOGAT activities by splicing the NADHbinding domain of NADH-GOGAT onto the Fd-GOGAT gene (see FIG. 2). Such achimeric GOGAT enzyme would have the advantage of being able to utilizeeither NADH or ferredoxin as a reductant in the GOGAT reaction. Theectopic expression of this new enzyme may result in more efficientsynthesis of glutamate. Another example of enzyme modification presentedherein (see Section 7.0) is the engineering of an AS enzyme which has adomain deleted to alter its substrate specificity.

In accordance to the present invention, controlling the tissue anddevelopmental expression patterns of the nitrogen assimilation orutilization enzymes may be important to achieving the desired plantimprovements. In instances where plants are engineered for ectopicoverexpression of the enzymes involved in the normal or alternativeammonia assimilation pathways, preferred embodiments of the presentinvention involve effecting altered expression in many or all parts ofthe plant. In instances where plants are engineered for ectopicoverexpression of enzymes catalyzing the use of assimilated nitrogen,preferred embodiments of the present invention limit such expressions tonitrogen "sink" tissues and structures such as leaves and seeds.

5.2. Generating Transgenic Plants 5.2.1. Nucleic Acid Constructs

The properties of the nucleic acid sequences are varied as are thegenetic structures of various potential host plant cells. The preferredembodiments of the present invention will describe a number of featureswhich an artisan may recognize as not being absolutely essential, butclearly advantageous. These include methods of isolation, synthesis orconstruction of gene constructs, the manipulations of the geneconstructs to be introduced into plant cells, certain features of thegene constructs, and certain features of the vectors associated with thegene constructs.

Further, the gene constructs of the present invention may be encoded onDNA or RNA molecules. According to the present invention, it ispreferred that the desired, stable genotypic change of the target plantbe effected through genomic integration of exogenously introducednucleic acid construct(s), particularly recombinant DNA constructs.Nonetheless, according to the present inventions, such genotypic changescan also be effected by the introduction of episomes (DNA or RNA) thatcan replicate autonomously and that are somatically and germinallystable. Where the introduced nucleic acid constructs comprise RNA, planttransformation or gene expression from such constructs may proceedthrough a DNA intermediate produced by reverse transcription.

The nucleic acid constructs described herein can be produced usingmethods well known to those skilled in the art. Artisans can refer tosources like Sambrook et al., 1989, Molecular Cloning: a laboratorymanual, Cold Spring Harbor Laboratory Press, Plainview, N.Y. forteachings of recombinant DNA methods that can be used to isolate,characterize, and manipulate the components of the constructs as well asto built the constructs themselves. In some instances, where the nucleicacid sequence of a desired component is known, it may be advantageous tosynthesize it rather than isolating it from a biological source. In suchinstances, an artisan can refer to teachings of the likes of Carutherset al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233, and of Chow andKempe, 1981, Nuc. Acids Res. 9:2807-2817. In other instances, thedesired components may be advantageously produced by polymerase chainreaction (PCR) amplification. For PCR teachings, an artisan can refer tothe like of Gelfand, 1989, PCR Technology, Principles and Applicationsfor DNA Amplification, H. A. Erlich, ed., Stockton Press, N.Y., CurrentProtocols In Molecular Biology, Vol. 2, Ch. 15, Ausubel et al. eds.,John Wiley & Sons, 1988.

5.2.1.1. Expression Constructs

In accordance to the present invention, a plant with ectopicoverexpression of a nitrogen assimilation or utilization enzyme may beengineered by transforming a plant cell with a gene construct comprisinga plant promoter operably associated with a sequence encoding thedesired enzyme. (Operably associated is used herein to mean thattranscription controlled by the "associated" promoter would produce afunctional messenger RNA, whose translation would produce the enzyme.)In a preferred embodiment of the present invention, the associatedpromoter is a strong and non tissue- or developmental-specific plantpromoter (e.g. a promoter that strongly expresses in many or all tissuetypes). Examples of such strong, "constitutive" promoters include, butare not limited to, the CaMV 35S promoter, the T-DNA mannopinesynthetase promoter, and their various derivatives.

In another embodiment of the present invention, it may be advantageousto engineer a plant with a gene construct operably associating a tissue-or developmental-specific promoter with a sequence encoding the desiredenzyme. For example, where expression in photosynthetic tissues andorgans are desired, promoters such as those of the ribulose bisphosphatecarboxylase (RUBISCO) genes or chlorophyll a/b binding protein (CAB)genes may be used; where expression in seed is desired, promoters suchas those of the various seed storage protein genes may be used; whereexpression in nitrogen fixing nodules is desired, promoters such thoseof the legehemoglobin or nodulin genes may be used; where root specificexpression is desired, promoters such as those encoding forroot-specific glutamine synthetase genes may be used (see Tingey et al.,1987, EMBO J. 6:1-9; Edwards et al., 1990, Proc. Nat. Acad. Sci. USA87:3459-3463).

In an additional embodiment of the present invention, it may beadvantageous to transform a plant with a gene construct operablyassociating an inducible promoter with a sequence encoding the desiredenzyme. Examples of such promoters are many and varied. They include,but are not limited to, those of the heat shock genes, the defenseresponsive gene (e.g., phenylalanine ammonia lyase genes), wound inducedgenes (e.g., hydroxyproline rich cell wall protein genes),chemically-inducible genes (e.g., nitrate reductase genes, gluconasegenes, chitinase genes, etc.), dark-inducible genes (e.g., asparaginesynthetase gene (Coruzzi and Tsai, U.S. Pat. No. 5,256,558, Oct. 26,1993, Gene Encoding Plant Asparagine Synthetase) to name just a few.

In yet another embodiment of the present invention, it may beadvantageous to transform a plant with a gene construct operably linkinga modified or artificial promoter to a sequence encoding the desiredenzyme. Typically, such promoters, constructed by recombining structuralelements of different promoters, have unique expression patterns and/orlevels not found in natural promoters. See e.g., Salina et al., 1992,Plant Cell 4:1485-1493, for examples of artificial promoters constructedfrom combining cis-regulatory elements with a promoter core.

In yet an additional embodiment of the present invention, the ectopicoverexpression of a nitrogen assimilation or utilization enzyme may beengineered by increasing the copy number of the gene encoding thedesired enzyme. One approach to producing a plant cell with increasedcopies of the desired gene is to transform with nucleic acid constructsthat contain multiple copies of the gene. Alternatively, a gene encodingthe desired enzyme can be placed in a nucleic acid construct containingan amplification-selectable marker (ASM) gene such as the glutaminesynthetase or dihydrofolate reductase gene. Cells transformed with suchconstructs is subjected to culturing regimes that select cell lines withincreased copies of ASM gene. See Donn et al., 1984, J. Mol. Appl.Genet. 2:549-562, for a selection protocol used to isolate of a plantcell line containing amplified copies of the GS gene. Because thedesired gene is closely linked to the ASM gene, cell lines thatamplified the ASM gene would also likely to have amplified the geneencoding the desired enzyme.

In one more embodiment of the present invention, the ectopicoverexpression of a nitrogen assimilation or utilization enzyme may beengineered by transforming a plant cell with nucleic acid constructencoding a regulatory gene that controls the expression of theendogenous gene or an transgene encoding the desired enzyme, wherein theintroduced regulatory gene is modified to allow for strong expression ofthe enzyme in the desired tissues and/or developmental stages.synthetase promoter, and their various derivatives.

5.2.1.2. Suppression Constructs

In accordance to the present invention, a desired plant may beengineered by suppressing GS activity or the activities of other enzymesin nitrogen assimilation/metabolism (FIG. 1). In an embodiment, thesuppression may be engineered by transforming a plant cell with a geneconstruct encoding an antisense RNA complementary to a segment or thewhole of a host target RNA transcript, including the mature target mRNA.In another embodiment, target gene (e.g., GS mRNA) suppression may beengineered by transforming a plant cell with a gene construct encoding aribozyme that cleaves a host target RNA transcript, (e.g., GS RNAtranscript, including the mature GS mRNA).

In yet another embodiment, target gene suppression may be engineered bytransforming a plant cell with a gene construct encoding the targetenzyme containing a "dominant negative" mutation. Preferred mutationsare those affecting catalysis, substrate binding (e.g., for GS, thebinding site of glutamate or ammonium ion), or product release. A usefulmutation may be a deletion or point-mutation of the critical residue(s)involved with the above-mentioned processes. An artisan can refer toteachings herein and of Herskowitz (Nature, 329:219-222, 1987) forapproaches and strategies to constructing dominant negative mutations.

For all of the aforementioned suppression constructs, it is preferredthat such gene constructs express with the same tissue and developmentalspecificity as the target gene. Thus, it is preferred that thesesuppression constructs be operatively associated with the promoter ofthe target gene. Alternatively, it may be preferred to have thesuppression constructs expressed constitutively. Thus, a strong,constitute promoter, such as the CaMV 35S promoter, may also be used toexpress the suppression constructs. A most preferred promoter for thesesuppression constructs is a modified promoter of the target gene,wherein the modification results in enhanced expression of the targetgene promoter without changes in the tissue or developmentalspecificities.

In accordance with the present invention, desired plants with suppressedtarget gene expression may also be engineered by transforming a plantcell with a co-suppression construct. A co-suppression constructcomprises a functional promoter operatively associated with a completeor partial coding sequence of the target gene. It is preferred that theoperatively associated promoter be a strong, constitutive promoter, suchas the CaMV 35S promoter. Alternatively, the co-suppression constructpromoter can be one that expresses with the same tissue anddevelopmental specificity as the target gene. Such alternative promoterscould include the promoter of the target gene itself (e.g., a GSpromoter to drive the expression of a GS co-suppression construct).

According to the present invention, it is preferred that theco-suppression construct encodes a incomplete target mRNA or defectivetarget enzyme, although a construct encoding a fully functional targetmRNA or enzyme may also be useful in effecting co-suppression.

In embodiments, where suppression of most, if not all, GS isozymes isdesired, it is preferred that the co-suppression construct encodes acomplete or partial copy of chloroplastic GS mRNA (e.g., pea GS2 mRNA).As disclosed herein (section 6.2.2.), such constructs are particularlyeffective in suppressing the expression of the target gene.

In accordance with the present invention, desired plants with suppressedtarget gene expression may also be engineered by transforming a plantcell with a construct that can effect site-directed mutagenesis of theendogenous target gene. (See Offringa et al., 1990, EMBO J. 9:3077-84;and Kanevskii et al., 1990, Dokl. Akad. Nauk. SSSR 312:1505-1507) fordiscussions of nucleic constructs for effecting site-directedmutagenesis of target genes in plants.) It is preferred that suchconstructs effect suppression of target gene by replacing the endogenoustarget gene sequence through homologous recombination with none orinactive coding sequence.

5.2.1.3. Other Features of Recombinant Nucleic Acid Constructs

The recombinant construct of the present invention may include aselectable marker for propagation of the construct. For example, aconstruct to be propagated in bacteria preferably contains an antibioticresistance gene, such as one that confers resistance to kanamycin,tetracycline, streptomycin, or chloramphenicol. Suitable vectors forpropagating the construct include plasmids, cosmids, bacteriophages orviruses, to name but a few.

In addition, the recombinant constructs may include plant-expressibleselectable or screenable marker genes for isolating, identifying ortracking of plant cells transformed by these constructs. Selectablemarkers include, but are not limited to, genes that confer antibioticresistances (e.g., resistance to kanamycin or hygromycin) or herbicideresistance (e.g., resistance to sulfonylurea, phosphinothricin, orglyphosate). Screenable markers include, but are not limited to, thegenes encoding β-glucuronidase (Jefferson, 1987, Plant Molec Biol. Rep5:387-405), luciferase (Ow et al., 1986, Science 234:856-859), B and C1gene products that regulate anthocyanin pigment production (Goff et al.,1990, EMBO J 9:2517-2522).

In embodiments of the present invention which utilize the Agrobacteriumsystem for transforming plants (see infra), the recombinant DNAconstructs additionally comprise at least the right T-DNA bordersequence flanking the DNA sequences to be transformed into plant cell.In preferred embodiments, the sequences to be transferred in flanked bythe right and left T-DNA border sequences. The proper design andconstruction of such T-DNA based transformation vectors are well knownto those skilled in the art.

5.2.2. Transformation of Plants and Plant Cells

According to the present invention, a desirable plant may be obtained bytransforming a plant cell with the nucleic acid constructs describedherein. In some instances, it may be desirable to engineer a plant orplant cell with several different gene constructs. Such engineering maybe accomplished by transforming a plant or plant cell with all of thedesired gene constructs simultaneously. Alternatively, the engineeringmay be carried out sequentially. That is, transforming with one geneconstruct, obtaining the desired transformant after selection andscreening, transforming the transformant with a second gene construct,and so on. In preferred embodiments each gene constructs would be linkedto a different selectable or screenable marker gene so as to facilitatethe identification of plant transformants containing multiple geneinserts. In another embodiment, several different genes may beincorporated into one plant by crossing parental lines engineered foreach gene.

In an embodiment of the present invention, Agrobacterium is employed tointroduce the gene constructs into plants. Such transformationspreferably use binary Agrobacterium T-DNA vectors (Bevan, 1984, Nuc.Acid Res. 12:8711-8721), and the co-cultivation procedure (Horsch etal., 1985, Science 227:1229-1231). Generally, the Agrobacteriumtransformation system is used to engineer dicotyledonous plants (Bevanet al., 1982, Ann. Rev. Genet 16:357-384; Rogers et al., 1986, MethodsEnzymol. 118:627-641). The Agrobacterium transformation system may alsobe used to transform as well as transfer DNA to monocotyledonous plantsand plant cells. (see Hernalsteen et al., 1984, EMBO J 3:3039-3041 ;Hooykass-Van Slogteren et al., 1984, Nature 311:763-764; Grimsley etal., 1987, Nature 325:1677-179; Boulton et al., 1989, Plant Mol. Biol.12:31-40.; Gould et al., 1991, Plant Physiol. 95:426-434).

In other embodiments, various alternative methods for introducingrecombinant nucleic acid constructs into plants and plant cells may alsobe utilized. These other methods are particularly useful where thetarget is a monocotyledonous plant or plant cell. Alternative genetransfer and transformation methods include, but are not limited to,protoplast transformation through calcium-, polyethylene glycol (PEG)-or electroporation-mediated uptake of naked DNA (see Paszkowski et al.,1984, EMBO J 3:2717-2722, Potrykus et al. 1985, Molec. Gen. Genet.199:169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828;Shimamoto, 1989, Nature 338:274-276) and electroporation of planttissues (D'Halluin et al., 1992, Plant Cell 4:1495-1505). Additionalmethods for plant cell transformation include microinjection, siliconcarbide mediated DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter9:415-418), and microprojectile bombardment (see Klein et al., 1988,Proc. Nat. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al., 1990, PlantCell 2:603-618).

According to the present invention, a wide variety of plants and plantcell systems may be engineered for the desired physiological andagronomic characteristics described herein using the nucleic acidconstructs of the instant invention and the various transformationmethods mentioned above. In preferred embodiments, target plants andplant cells for engineering include, but are not limited to, those ofmaize, wheat, rice, soybean, tomato, tobacco, carrots, potato, sugarbeets, sunflower, yam, Arabidopsis, rape seed, and petunia.

5.2.3. Selection and Identification of Transformed Plants and PlantCells

According to the present invention, desired plants may be obtained byengineering the disclosed gene constructs into a variety of plant celltypes, including but not limited to, protoplasts, tissue culture cells,tissue and organ explants, pollens, embryos as well as whole plants. Inan embodiment of the present invention, the engineered plant material isselected or screened for transformants (those that have incorporated orintegrated the introduced gene construct(s)) following the approachesand methods described below. An isolated transformant may then beregenerated into a plant. Alternatively, the engineered plant materialmay be regenerated into a plant or plantlet before subjecting thederived plant or plantlet to selection or screening for the marker genetraits. Procedures for regenerating plants from plant cells, tissues ororgans, either before or after selecting or screening for markergene(s), are well known to those skilled in the art.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells may also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs of the present invention. Suchselection and screening methodologies are well known to those skilled inthe art.

Physical and biochemical methods also may be also to identify plant orplant cell transformants containing the gene constructs of the presentinvention. These methods include but are not limited to: 1) Southernanalysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

5.2.4. Screening of Transformed Plants for Those With Improved AgronomicTraits

According to the present invention, to obtain plants with improvedagronomic characteristics, the transformed plants may be screened forthose exhibiting the desired physiological alteration. For example,where the plants have been engineered for ectopic overexpression of a GSenzyme, transformed plants are examined for those expressing the GSenzyme at the desired level and in the desired tissues and developmentalstages. Where the plants have been engineered for suppression of atarget gene, transformed plants are examined for those expressing thetarget gene product (e.g., RNA or protein) at reduced levels in varioustissues. The plants exhibiting the desired physiological changes, e.g.,ectopic GS overexpression or GS suppression, may then be subsequentlyscreened for those plants that have the desired agronomic changes.

Alternatively, the transformed plants may be directly screened for thoseexhibiting the desired agronomic changes. In one embodiment, suchscreening may be for productive growth of the transformed plants undernitrogen nutrient deficient conditions. That is screen for growth oftransformed plants under conditions, with respect to the availablenitrogen nutrient, that cause the growth of wild-type plant to cease orto be so diminished as to significantly reduce the size or quality ofthe wild-type plant. An example of a nitrogen nutrient deficientcondition for tobacco and plants with similar nitrogen nutrientrequirements is that where the sole nitrogen nutrient in the soil orsynthetic medium is (a) nitrate supplied or periodically applied at aconcentration of 0.5 mM or lower, or (b) physiological equivalents ofnitrate (e.g., ammonium or a mix of nitrate and ammonium) supplied orperiodically applied at a concentration that is physiologicallyequivalent to 0.5 mM nitrate or lower (see Eckes et al., 1988,Australian Patent Office document no. AU-A-17321/88). Another example ofa nitrogen nutrient deficient condition is that where the steady statelevel of the available nitrogen nutrient in the soil or synthetic mediumis less than about 0.02 mM nitrate or physiological equivalents thereof.The term nitrate as used herein means any one or any mix of the nitratesalts commonly used as plant nitrogen fertilizer, e.g., potassiumnitrate, calcium nitrate, sodium nitrate, ammonium nitrate, etc. Theterm ammonium as used herein means any one or any mix of the ammoniumsalts commonly used as plant nitrogen fertilizer, e.g., ammoniumnitrate, ammonium chloride, ammonium sulfate, etc.

In other embodiments, the screening of the transformed plants may be forimproved agronomic characteristics (e.g., faster growth, greatervegetative or reproductive yields, or improved protein contents, etc.),as compared to unengineered progenitor plants, when cultivated undernitrogen non-limiting growth conditions (i.e., cultivated using soils ormedia containing or receiving sufficient amounts of nitrogen nutrientsto sustain healthy plant growth). An example of nitrogen non-limitingconditions for tobacco and plants with similar nitrogen nutrientrequirements is that where the sole nitrogen nutrient in soil orsynthetic medium is (a) nitrate supplied or periodically applied at aconcentration of 10 mM or higher, or (b) physiological equivalents ofnitrate supplied or periodically applied at a concentration that isphysiologically equivalent to 10 mM nitrate or higher. Another exampleof nitrogen non-limiting conditions is that where the steady state levelof the available nitrogen nutrient in the soil or synthetic medium is atleast about 1.0 mM potassium nitrate or physiological equivalentsthereof. Additional guidance with respect to what are nitrogen nutrientdeficient or "non-limiting" conditions for plant growth may be found inthe art. See for example, Hewitt, E. J., Sand and Water Culture MethodsUsed in the Study of Plant Nutrition, 2nd ed., Farnham Royal (Bucks),Commonwealth Agricultural Bureaux, 1966; and Hewitt, E. J., PlantMineral Nutrition, London, English University Press, 1975.

In embodiments where the transformed plants are legumes, directscreenings for transformed plants with the desired agronomic changes andimprovements may be conducted as described above but under conditionswhere nodule formation or nitrogen-fixation is suppressed.

According to the present invention, plants engineered with thealterations in nitrogen assimilation or utilization processes mayexhibit improved nitrogen contents, altered amino acid or proteincompositions, vigorous growth characteristics, increased vegetativeyields or better seed yields and qualities. Engineered plants and plantlines possessing such improved agronomic characteristics may beidentified by examining any of following parameters: 1) the rate ofgrowth, measured in terms of rate of increase in fresh or dry weight; 2)vegetative yield of the mature plant, in terms of fresh or dry weight;3) the seed or fruit yield; 4) the seed or fruit weight; 5) the totalnitrogen content of the plant; 6) the total nitrogen content of thefruit or seed; 7) the free amino acid content of the plant; 8) the freeamino acid content of the fruit or seed; 9) the total protein content ofthe plant; and 10) the total protein content of the fruit or seed. Theprocedures and methods for examining these parameters are well known tothose skilled in the art.

According to the present invention, a desired plant is one that exhibitsimprovement over the control plant (i.e., progenitor plant) in one ormore of the aforementioned parameters. In an embodiment, a desired plantis one that shows at least 5% increase over the control plant in atleast one parameter. In a preferred embodiment, a desired plant is onethat shows at least 20% increase over the control plant in at least oneparameter. Most preferred is a plant that shows at least 50% increase inat least one parameter.

5.3. Utility of the Invention

The engineered plants of the present invention may be productivelycultivated under nitrogen nutrient deficient conditions (i.e.,nitrogen-poor soils and low nitrogen fertilizer inputs) that would causethe growth of wild-type plants to cease or to be so diminished as tomake the wild-type plants practically useless. The engineered plantsalso may be advantageously used to achieve earlier maturing, fastergrowing, and/or higher yielding crops and/or produce more nutritiousfoods and animal feedstocks when cultivated using nitrogen non-limitinggrowth conditions (i.e., soils or media containing or receivingsufficient amounts of nitrogen nutrients to sustain healthy plantgrowth). Nitrogen non-limiting growth conditions vary between speciesand for varieties within a species. However, one skilled in the artknows what constitute nitrogen non-limiting growth conditions for thecultivation of most, if not all, important crop and ornamental plants.For example, for the cultivation of wheat see Alcoz et al., AgronomyJournal 85:1198-1203 (1993), Rao and Dao, J. Am. Soc. Agronomy84:1028-1032 (1992), Howard and Lessman, Agronomy Journal 83:208-211(1991); for the cultivation of corn see Tollenear et al., AgronomyJournal 85:251-255 (1993), Straw et al., Tennessee Farm and HomeScience: Progress Report, 166:20-24 (Spring 1993), Miles, S. R., J. Am.Soc. Agronomy 26:129-137 (1934), Dara et al., J. Am. Soc. Agronomy84:1006-1010 (1992), Binford et al., Agronomy Journal 84:53-59 (1992);for the cultivation of soybean see Chen, et al., Canadian Journal ofPlant Science 72:1049-1056 (1992), Wallace et al. Journal of PlantNutrition 13:1523-1537 (1990); for the cultivation of rice see Oritaniand Yoshida, Japanese Journal of Crop Science 53:204-212 (1984); for thecultivation of linseed see Diepenbrock and Porksen, Industrial Crops andProducts 1:165-173 (1992); for the cultivation of tomato see Grubingeret al., Journal of the American Society for Horticultural Science118:212-216 (1993), Cerne, M., Acta Horticulture 277:179-182, (1990);for the cultivation of pineapple see Magistad et al. J. Am. Soc.Agronomy 24:610-622 (1932), Asoegwu, S. N., Fertilizer Research15:203-210 (1988), Asoegwu, S. N., Fruits 42:505-509 (1987), for thecultivation of lettuce see Richardson and Hardgrave, Journal of theScience of Food and Agriculture 59:345-349 (1992); for the cultivationof mint see Munsi, P. S., Acta Horticulturae 306:436-443 (1992); for thecultivation of camomile see Letchamo, W., Acta Horticulturae 306:375-384(1992); for the cultivation of tobacco see Sisson et al., Crop Science31:1615-1620 (1991); for the cultivation of potato see Porter andSisson, American Potato Journal, 68:493-505 (1991); for the cultivationof brassica crops see Rahn et al., Conference "Proceedings, secondcongress of the European Society for Agronomy"Warwick Univ., p.424-425(Aug. 23-28, 1992); for the cultivation of banana see Hegde andSrinivas, Tropical Agriculture 68:331-334 (1991), Langenegger and Smith,Fruits 43:639-643 (1988); for the cultivation of strawberries see Humanand Kotze, Communications in Soil Science and Plant Analysis 21:771-782(1990); for the cultivation of songhum see Mahalle and Seth, IndianJournal of Agricultural Sciences 59:395-397 (1989); for the cultivationof plantain see Anjorin and Obigbesan, Conference "InternationalCooperation for Effective Plantain and Banana Research" Proceedings ofthe third meeting. Abidjan, Ivory Coast, p. 115-117 (May 27-31, 1985);for the cultivation of sugar can e see Yadav, R. L., Fertiliser News31:17-22 (1986), Yadav and Sharma, Indian Journal of AgriculturalSciences 53:38-43 (1983); for the cultivation of sugar beet see Draycottet al. , Conference "Symposium Nitrogen and Sugar Beet" InternationalInstitute for Sugar Beet Research--Brussels Belgium, p. 293-303 (1983).See also Goh and Haynes, "Nitrogen and Agronomic Practice" in MineralNitrogen in the Plant-Soil System, Academic Press, Inc., Orlando, Fla.,p. 379-468 (1986), Engelstad, O. P., Fertilizer Technology and Use,Third Edition, Soil Science Society of America, p.633 (1985), Yadav andSharmna, Indian Journal of Agricultural Sciences, 53:3-43 (1983).

GS suppression have utility in that some GS suppressed plants,particularly legumes, may grow faster or have higher nitrogen contentsthan non-suppressed plants. (See Knight and Langston-Unkefer , Science241:951-954). GS suppressed plants may also have altered amino acid orprotein contents, making such plants useful in preparation of specialdietary foods. Further, all the engineered plants disclosed herein mayalso serve as breeding stocks for developing agriculturally useful plantlines.

6. EXAMPLE

Ectopic Overexpression of Glutamine Synthetase in Plants Causes anIncrease in Plant Growth Phenotype

Described herein is a molecular-genetic approach to manipulate nitrogenuse efficiency in transgenic plants. The approach relies on the ectopicexpression of glutamine synthetase, that express GS in cell-types and/orat levels which the GS expression is not normally found. The pattern ofcell-specific GS expression in transgenic plants is altered byconstitutively overexpressing the cytosolic GS (which is normally onlyexpressed in phloem) in all cell-types. Such ectopic expression of GSmay circumvent physiological limitations which result from thecompartmentalization and cell-type specificity of nitrogen assimilatoryenzymes. The ectopic high-level expression of cytosolic GS in mesophyllcells might provide an alternate route for the reassimilation of ammonialost via photorespiration. This may provide a growth advantage as theamount of ammonia lost via photorespiration exceeds primary nitrogenassimilation by 10-fold (Wallsgrove et al., 1983, Plant Cell Environ.6:301-309; Keys et al., 1978, Nature, 275:741-743). The studiesdisclosed herein show that constitutive overexpression of a heterologousGS subunit for cytosolic GS leads to increases in GS mRNA, GS protein,total GS activity, native GS holoenzyme, and, in one case, to theproduction of a novel GS holoenzyme. Transformed plants whichoverexpress cytosolic GS have a statistically significant growthadvantage compared to wild type. They grow faster, attain a higher finalfresh weight and have more soluble proteins than untransformedprogenitor plants during the vegetative stage of their development. Insome instances, however, overexpression of cytosolic GS and/orchloroplastic GS leads to a down regulation of endogenous geneexpression or co-suppression. Some transformed plants containingcytosolic GS overexpression constructs and all transformed plantscontaining chloroplastic GS2 constructs do not overexpress GS, butrather are suppressed for GS expression, including suppression of theendogenous GS gene (i.e., co-suppression). Such GS co-suppressed plantsmay show poorer growth characteristics, but may have altered amino acidand protein contents due to shunting of nitrogen into other nitrogenassimilation/metabolism pathways.

6.1. Material and Methods 6.1.1. Plant Expression Vector Construction

Plant expression vectors pTEV 4,5,7, and 8 were constructed as follows.A HindIII-EcoRI fragment containing the 35S promoter from the Strasbourgstrain of the Cauliflower mosaic virus (CaMV) extending from -941 to +26relative to the start of transcription was inserted into pBluescript KSII-(pT109) (Hohn et al., 1982, Curr. Topics Microbiol. Immunol.96:194-236). The polylinker sequence between the HindIII and XhoI siteswas then modified to include Xbal, SstI, and StuI sites (pT145). Thisenabled a T4 polymerase-treated SstI-EcoRI fragment derived from pBIN19(Clontech) and containing the nopaline synthase transcriptionalterminator to be inserted at the StuI site creating pT161. Theexpression cassette thus constructed was flanked by EcoRI sites and wastransferred to pW3, a plasmid derived from pBIN19 (Bevan, 1984, NucleicAcids Res. 12:8711-8721) containing a modified polylinker. A cloneoriented with the 5' end of the promoter adjacent to the left border ofpW3 was selected (pW63) and numerous cloning sites were inserted betweenpromoter and terminator. This created the following binary vectors withthe unique cloning sites listed (FIG. 3): pTEV4(HindIII-XbaI-BamHI-XhoI), pTEV5 (HindIII-StuI-SstI-KpnI), pTEV8(HindIII-XhoI-BamHI-Xbal), pTEV9 (HindIII-KpnI-SstI-StuI).

6.1.2. Transfer of GS cDNAs to Binary Expression Vectors

cDNAs corresponding to the pea genes for cytosolic GS1 and GS3A, andchloroplastic GS2 were transferred from pBluescript to the binaryexpression vectors described above (see FIG. 4). These cDNAs havepreviously been described as GS299, GS341, and GS185 respectively(Tingey et al., 1987, EMBO J. 6:1-9; Tingey et al., 1988, J. Biol. Chem.263:9651:9657). For chloroplastic GS2, a modified cDNA was constructedwhich incorporated the first intron of the genomic sequence into thecDNA at the appropriate position (Z54). This was made using thepolymerase chain reaction to amplify a fragment extending from the 5'end of the cDNA to the BsmI site located within exon 2 (at amino acid43), which could then be cloned into the cDNA in pBluescript. Forcytosolic GS3A a modified cDNA (Z17) was constructed by exchange-cloninga BgIII-KpnI fragment from a genomic GS3A clone into the pBluescriptcDNA clone generating a cDNA sequence into which all genomic introns(from amino acid 6 onwards) had been inserted. The purpose ofconstructing cDNA incorporating introns was to attempt to enhanceexpression in transgenic plants as has been shown in monocots (Sinibaldiand Mettler, 1991). The cDNAs were transferred from pBluescript to thefollowing binary expression vectors: GS1-pTEV4 into XbaI-XhoI sites tofour pZ3 (NRRL Accession No. B-21330); GS3A and modified GS3A-pTEV4 intoXbaI-XhoI sites to form, respectively, pZ9 (NRRL Accession No. B-21331)and pZ17 (NRRL Accession No. 21332); GS2 and modified GS2-pTEV5 intoStuI-KpnI sites to form respectively, pZ41 (NRRL Accession No. B-21333)and pZ54 (NRRL Accession No. B-31334).

6.1.3. Plant Transformations

Binary vector constructions were transferred into the disarmedAgrobacterium strain LBA4404 by triparental mating using a previouslydescribed procedure (Bevan, 1984, Nucleic Acids Res. 12:8711-8721).Nicotiana tabacum line SR1 was transformed by a leaf inoculationprocedure (Horsch et al., 1985, Science 227--1299-1231), and regeneratedshoots were selected on medium containing 200 μg/ml kanamycin. Primarytransformants were maintained in sterile culture and subsequently grownto maturity in soil. Transgenic seeds were sterilized in 10% sodiumhypochlorite and germinated on medium containing 100 μg/ml kanamycin.

6.1.4. GS Protein and Enzyme Activity Analysis

Soluble proteins were extracted from tobacco and pea leaf tissue aspreviously described (Tingey and Coruzzi, 1987, Plant Physiol.84:366-373). Proteins were denatured and separated in 12% acrylamide bySDS-PAGE and electroblotted onto nitrocellulose. Western analysis wasundertaken using the ProtoBlot kit supplied by Promega and a mixture ofantibodies raised to tobacco chloroplast GS2 and Phaseolus cytosolic GS(Hirel et al., 1984, Plant Physiol. 74:448-450; Lara et al., 1984, PlantPhysiol. 76:1019-1023). Total GS activity in transformants wasdetermined using a previously described ADP-dependent transferase assay(Shapiro and Stadtman, 1970, Methods Enzymol. 17A;910-922).Non-denaturing gel electrophoresis followed a published protocol (Davis,1964, Annals New York Acad. Sci. 121:404-427) in conjunction with theADP-dependent transferase assay for GS isozyme detection.

6.1.5. RNA Analysis

RNA was isolated using "RNA matrix" from Bio101 following the protocolsuggested by the manufacturer. Total RNA was electrophoresed in 40 mMtriethanolamine, 2 mM EDTA and 3.2% formaldehyde in 1.2% agarose(Thomas, 1983, Methods Enzymol. 100:255-266). Gels were soaked in 10 mMsodium phosphate and capillary blotted onto Hybond-N nylon membrane(Amersham). cDNAs were labelled either using the random primer plusextension reagent labeling system supplied by NEN, and strand specificriboprobes were made using the Stratagene RNA transcription kit. Aqueoushybridizations were done according to the membrane manufacturer'sprotocol, and blots were washed in 0.1×SSPE, 0.1%×SDS.

6.1.6. Plant Growth Conditions

Progenies of primary transformants previously characterized asexpressing GS1 or GS3A at high levels were germinated on Murashige-Skoog(MS) medium containing 100 μg/ml kanamycin. After 14 days kanamycinresistant seedlings were transferred to 4 inch pots filled with whitesand, which were covered with Saran Wrap™ for approximately one week toprevent excessive transpiration and enable seedlings to becomeestablished. Pots were irrigated periodically with an excess of 1XHoagland's solution containing 10 mM potassium nitrate as the onlynitrogen source. Subsequently between three and seven plants weresacrificed for fresh weight determination each week, continuing for aperiod of four weeks until shading of neighbors was apparent. Plantswere grown under a light-dark cycle of 16-8 h with a temperature cycleof 24-18° C. Daytime light intensity was 1000 lux.

6.2. Results 6.2.1. GS Constructions Introduced into Transgenic Plants

Pisum sativum cDNAs for chloroplastic GS2 (aka GS185 (Tingey et al.,1988, J. Biol. Chem. 263:9651-9657)), cytosolic GS1 (aka GS299 (Tingeyet al., 1988, J. Biol. Chem. 263:9651-9657)) and GS3A (aka GS341 (Tingeyet al., 1987, EMBO J. 6:1-9)) were inserted into pTEV binary expressionvectors (see FIGS. 3 and 4) for expression behind the CaMV 35S promoterand transferred to transgenic tobacco. For GS2 (construct Z54, FIG. 4)and GS3A (construct Z17, FIG. 4) cDNAs incorporating one or more intronswere constructed and expressed behind the CaMV 35S promoter. The purposeof constructing cDNAs incorporating introns was to attempt to enhanceexpression in transgenic plants, as has been shown for monocots(Sinibaldi and Mettler, 1991, Progress in Nucleic Acid Research andMolecular Biology 42:1991). In addition, unmodified full-length GS cDNAswere also expressed under the 35S-CaMV promoter for GS2 (Z41), G3A (Z9),and GS1 (Z3) (see FIG. 4). For each of the 35S-CaMV-GS constructionsdetailed in FIG. 4, at least eight primary (T1) transformants wereanalyzed and representative samples are shown in FIG. 5. For selectedprimary transformants, four kanamycin-resistant T2 progeny plants werealso analyzed (FIG. 6). The analysis of T1 and T2 plants presented belowincludes Western analysis (FIG. 5 and FIG. 6, panel A); Northern blotanalysis (FIG. 6, panel B), GS holoenzyme analysis (FIG. 6, panel C),and GS enzyme activity analysis (FIG. 6, panel C and Tables 1A and 1B)and are representative of all the transgenic lines analyzed.

                  TABLE 1A                                                        ______________________________________                                        Total GS Activity in Primary Transformants (T1)                               Z41: 35S-GS2       Z54:35S-GS2 (modified)                                     ______________________________________                                        Z41-6      42          Z54-1      13                                          Z41-7      74          Z54-2      11                                          Z41-8      23          Z54-3      49                                          Z41-12     66          Z54-4      22                                          Z41-14     44          Z54-6      39                                          Z41-15     nd          Z54-7      25                                          Z41-16     65          Z54-8      23                                          Z41-18     29          Z54-9      25                                          Z41-20     35          Z54-10     33                                          Z41-23     76                                                                 Z41-24     32                                                                 Z41-25     67                                                                 Z41-27     29                                                                 Z41-32     22                                                                 Z41-33     85                                                                 Z17: 35S-GS3 (modified)                                                                          Z3: 35S-GS1                                                ______________________________________                                        Z17-3      138         Z3-1       nd                                          Z17-6      127         Z3-2       nd                                          Z17-7      119                                                                Z17-8       36                                                                Z17-9       45                                                                Z17-10      52                                                                Z17-12      28                                                                Z17-14     145                                                                ______________________________________                                         Total GS activity was determined for primary transformants and are            expressed as percentages compared to SR1 wildtype (=100).                     nd  not determined.                                                      

                  TABLE 1B                                                        ______________________________________                                        Total GS Activity in Primary Transformants                                    (T1) and their Progenies (T2)                                                 T1         T2-mean   T2-A    -B    -C    -D                                   ______________________________________                                        Z41: 35S-GS2                                                                  Z41-15 nd      27        15    7     75    11                                 Z41-20 35      50        53    33    31    81                                 Z41-33 85      35        31    30    32    46                                 Z54: 35S-GS2 (modified)                                                       Z54-2  11      28        30    19    21    42                                 Z54-7  25      22        29    21    18    19                                 Z54-8  23      35        34    39    31    35                                 Z17: 35S-GS3A (modified)                                                      Z17-6  127     100       112   99    94    96                                 Z17-7  119     107       104   103   111   108                                Z17-9  45      44        126   14    26    11                                 Z17-10 52      27        33    50    18    5                                  Z17-12 28      18        21    18    22    10                                 Z3: 35S-GS1                                                                   Z3-1   nd      123       108   129   113   140                                Z3-2   nd      120       114   129   121   116                                ______________________________________                                         Total GS activity was determined for primary transformants and four T2        progeny plants (labeled AD). Activity is expressed in percentage of SR1       wildtype (=100). nd = not determined.                                    

6.2.2. Analysis of Transgenic Plants Carrying 35S-Chloroplastic GS2 GeneFusions

Transgenic plants containing either of the 35S-GS2 constructs (Z41 orZ54; FIG. 4) were analyzed. Both the 35S-GS2 (Z41) and the modified(intron-containing) 35S-GS2 construct (Z54) gave similar results forboth primary T1 transformants and for T2 progeny plants. Western blotanalysis of all primary transformants revealed a significant reductionin the abundance of chloroplastic GS2 polypeptide (ctGS) (FIG. 5 lanes3-6), when compared to wild-type tobacco (FIG.

5, lane TL). Since the polyclonal GS2 antibodies have been shown torecognize both pea and tobacco GS2 (Tingey and Coruzzi, 1987, PlantPhysiol. 84:366-373; Tingey et al., 1988, J. Biol. Chem. 263:9651-9657)this reduction reflects a down-regulation of both the host tobacco GS2gene and also of the pea GS2 transgene. No change in the abundance ofthe cytosolic GS polypeptides (cyGS) was observed in these transformants(FIG. 5, lanes 3-6) compared to control untransformed wild-type tobacco(FIG. 5, lane TL). For Z41, all fourteen independent primarytransformants were down-regulated for total GS activity, with a high of85% wild-type activity to a low of 22% wild-type GS activity (Tables 1Aand 1B). For the Z54 constructs, all nine independent primarytransformants regenerated were down-regulated to below 50% of wild-typeGS activity, with a range of 49% to 11% (Tables 1A and 1B). From thesedata, it is apparent that the intron containing Z54 constructs wereseverely co-suppressed. By contrast, the Z41 construct was lessefficient at down-regulating endogenous tobacco chloroplastic GS2 andthese plants showed a wider range of co-suppression phenotypes (seevariation in GS activity amongst Z41 individuals in Tables 1A and 1B).Typically, plants co-suppressed for GS2 (Z54 or Z41) grew more slowlythan wild-type and developed intervenial chlorosis (see FIG. 10) dueeither to the toxicity associated with ammonia accumulation duringphotorespiration, or glutamine deficiency. These transformants weretherefore similar to the previously described GS2 mutants of barley(Wallsgrove et al., 1987, Plant Physiol. 83:155-158). Co-suppressedplants of either Z41 or Z54 type grown in an atmosphere of elevated(1.2%) CO₂ (to suppress photorespiration), or supplemented withglutamine, showed less severe symptoms, also supporting the conclusionthat these plants were deficient in GS2.

Four kanamycin-resistant T2 progeny plants from primary Z41 and Z54transformants were also analyzed (FIG. 6). The results obtained fromWestern analysis and for total GS activity for progenies were similar tothose observed for primary transformants (FIG. 6, panel A, and Table1B). FIG. 6 shows data for one representative T2 progeny member forseveral Z54 or Z41 primary transformants (FIG. 6, lanes 9-14). Westernblot analysis of these plants confirmed the low abundance of thechloroplast GS2 protein (FIG. 6, panel A) and non-denaturing GS activitygel analyses confirmed the reduced abundance of the GS2 holoenzyme (FIG.6, panel C, lanes 9-14) compared to wild-type tobacco (FIG. 6, panel C,lane TL). Northern analysis showed that transcripts from the GS2transgene were undetectable (FIG. 6, panel B, lanes 9-14) compared tothat present in control pea RNA (FIG. 6, panel B, lane P). These resultssuggest the specific co-suppression of tobacco chloroplastic GS2 fromthe insertion of a pea GS2 transgene. In addition, the pea GS2 transgenewas also silenced. Levels of cytosolic GS mRNA and protein wereunaffected in these GS2 co-suppressed plants.

6.2.3. Analysis of Transgenic Plants Carrying 35-S Cytosolic GS3A GeneFusions

Transgenic plants containing either type of 35S-GS3A construct (Z17 orZ9; FIG. 4) were analyzed. For Z17 (the intron containing line), of thethirteen independent primary transformants analyzed for GS activity, sixshowed overexpression of GS activity (119-145%) while seven showedco-suppression (52-28%) compared to untransformed controls (100%)(Tables 1A and 1B). FIGS. 5 and 6 contain data for representativeoverexpressers and co-suppressed lines of Z17. Transformant Z17-12 isco-suppressed for GS enzyme activity (27% of wild-type) and bothchloroplastic GS2 and cytosolic GS proteins are low (FIG. 5, lane 2)compared to wild-type tobacco (FIG. 5, lane TL). By contrast,transformant Z17-6 has elevated levels of total GS activity (127%) andincreased levels of cytosolic GS protein (FIG. 5, lane 1) compared towild-type tobacco (FIG. 5, lane TL). Analysis of the T2 progeny of otherindependent transformants revealed additional transformants to bedown-regulated for cytosolic GS protein (Z17-9B and Z17-10; FIG. 6,Panel A, lanes 6 and 7), while others had elevated levels of cytosolicGS (Z17-7 and Z17-9A; FIG. 6, Panel A, lanes 4 and 5). Theco-suppression phenomenon observed for the Z17 plants (Z17-9B, Z17-10,and Z17-12) is clearly different to that observed for the GS2transformants (Z54 and Z41) in that both chloroplastic GS2 and cytosolicGS are down-regulated in the GS3A co-suppressed plants (cf. FIG. 6,panel A, lanes 6-8 with lanes 9-14). FIG. 6 shows that co-suppressioncaused by 35S-GS3A (Z17-9B, Z17-10, Z17-12) is accompanied by reduced GSabundance (from Western and GS activity gel analysis; FIG. 6, panels Aand C, lanes 6-8) and virtually undetectable transcription of the GS3Atransgene (from Northern analysis; FIG. 6, panel B, lanes 6-8). Intransformants overexpressing the GS3A construct (Z17-6, Z17-7, andZ17-9A), the GS3A transcript is very abundant (FIG. 6, panel B, lanes3-5) and this reflects the greater abundance of cytosolic GS detectableby Western blot analysis (FIG. 6, panel A, lanes 3-5) and GS activityassays (Table 1). Non-denaturing GS activity gel analysis of solubleproteins from these Z17 transformants which overexpress cytosolic GS3Aindicates the existence of a novel GS holoenzyme (band A*, FIG. 6, panelC, lanes 3-5) which migrates more slowly than the predominantchloroplast GS2 holoenzyme in wild-type tobacco leaves (band B, FIG. 6,panel C, lane T). It is interesting that individual Z17 transformantscarrying the same GS3A transgene construction should give two distinctphenotypes, one of co-suppression (FIG. 6, lanes 6-8) and one ofoverexpression (FIG. 6, lanes 3-5).

To enlarge the size of the population of transgenic plants analyzed, asecond round of transformations was performed and yielded resultssimilar to those described above. Of a total of twenty-three independentprimary Z17 transformants analyzed, five were co-suppressed for GS andeight overexpressed GS. In addition, primary transformants were analyzedwhich contained an unmodified (intron-less) GS3A cDNA (Z9, FIG. 4); ofthe four Z9 primary transformants analyzed, one was co-suppressed for GSand two overexpressed cytosolic GS. This suggested no qualitativedifference between the Z17 (intron containing 35S-GS3A) and Z9 (35S-GS3AcDNA) constructions. Particularly intriguing is the observation thatZ17-9A and Z17-9B (FIG. 6, lanes 5 and 6) should have diverse phenotypesas these two T2 plants were derived by self-pollination from a singleprimary transformant. The Z17-9 primary transformant had been analyzedfor total GS activity and found to have reduced activity and thereforeto be co-suppressed (see Table 1). Two other T2 progeny plants of Z17-9were analyzed (Z17-9C and Z17-9D) and these were both found to beco-suppressed giving a ratio of 3:1 in favor of co-suppression in thispopulation.

6.2.4. Analysis of Transgenic Plants Carrying the 35S-Cytosolic GS1 GeneFusion

Transgenic plants containing the 35S-GS1 construct (Z3; see FIG. 4) werealso analyzed. Of the eight independent Z3 primary transformants, fivegave a clear phenotype of overexpression from Western and Northern blotanalysis, and none were co-suppressed. The T2 progeny of two of these Z3transformants are shown in FIG. 6. Both Z3-1 and Z3-2 show an increasedabundance of cytosolic GS protein (FIG. 6, panel A, lanes 1 and 2) andthis is reflected by the increased levels of GS mRNA (FIG. 6, panel B,lanes 1 and 2). Non-denaturing activity gel analysis demonstrated a GSholoenzyme (band C) (FIG. 6, panel C, lanes 1 and 2) which migratedfaster than the chloroplastic GS2 holoenzyme of tobacco leaves (FIG. 6,panel C, lane T). This faster migrating GS holoenzyme (band C) in the Z3plants corresponds in size to native pea cytosolic GS.

6.2.5. Analysis of Native and Novel Cytosolic GS Holoenzymes inTransgenic Plants

Ectopic expression of cytosolic GS3A (Z17) and GS1 (Z3) gave additional,but different, GS holoenzyme activity bands (e.g., bands A* and C)compared to chloroplast GS2 (band B) seen in wild-type tobacco leaves(FIG. 6, panel C). Electrophoresis of extracts from these transgenicplants was repeated in non-denaturing activity gels including forcomparison, lanes of pea root (PR) and tobacco root (TR) protein whichare enriched for the cytosolic GS holoenzyme (band C) FIG. 7A, lanes 2and 4), and extracts derived from purified pea chloroplasts (PC) andtobacco chloroplasts (TC) which are enriched for chloroplastic GS3holoenzyme (band B) (FIG. 7A, lanes 1 and 3). The additional GS1holoenzyme activity (band C) seen in extracts of leaves from transgenictobacco Z3-1 (FIG. 7A, lane 6) co-migrates with the native pea cytosolicGS band (band C, FIG. 7A, lanes 2 and 4). By contrast, the novel GS3Aactivity (band A*) seen in leaves of the Z17-7 transgenic plants (FIG.7A, lane 5) co-migrates with neither the cytosolic GS (band C) nor thechloroplastic GS2 band (band B) and is larger in size and more acidic incharge. To determine the subunit composition of the GS activity bandsA*, B, and C, these bands were excised from preparative gels, and theextracted proteins were reloaded on a denaturing SDS gel followed byWestern blot analysis for GS subunits (FIG. 7B). This analysis revealedthat both GS activity band A* and band C are comprised exclusively ofcytosolic GS polypeptides (FIG. 7B, lanes 2 and 4). This findingdiscounted the possibility that the larger GS3A activity band A* was theresult of the assembly of heterologous GS3A cytosolic subunits withendogenous tobacco pre-chloroplastic GS2 subunits. It is possible thatGS activity band A* represents the association of transgenic GS3Asubunits with a chaperonin-type protein, but attempts to dissociate sucha complex with ATP were unsuccessful. Consequently, the nature of thenovel GS holoenzyme remains unclear.

6.2.6. Selection of Transformants Ectopically Overexpressing CytosolicGS1 or GS3A for Growth Analysis

Two sets of plants which ectopically overexpress cytosolic GS3A (Z17) orcytosolic GS1 (Z3) were selected for growth analysis. From the firstround of transformations (see Experiment A, infra) plants Z3-1 and Z3-2were selected as GS1 high level expressers (FIG. 6, lanes 1 and 2; FIG.8, lanes 1 and 2), and plants Z17-6 and Z17-7 were selected as GS3A highlevel expressers (FIG. 5, lane 1; FIG. 6, lanes 3 and 4; FIG. 8, lanes 3and 4). Kanamycin resistant T2 progenies of these transformants wereselected for growth analysis in experiment A described below. From thesecond round of transformations, two more independently transformedGS1-overexpressing plants (Z3-3 and Z3-4); (FIG. 8, lanes 5 and 6), andtwo more independently transformed GS3A-overexpressing plants (Z17-3 andZ17-11) (FIG. 8, lanes 7 and 8) were selected for analysis. Thekanamycin-resistant T2 progenies of these plants were analyzed in thesecond growth experiment (Experiment B, infra).

6.2.7. Design of Plant Growth Experiments

Plant growth analysis was undertaken on the T2 progeny plants analyzedfor GS protein and RNA in FIG. 8. Individual T2 plants were grown inwhite sand and growth was assessed by fresh weight determination of 4-7plants per time point. Fresh weight measurements were taken only duringthe vegetative stage of growth when plants were growing rapidly and wereexclusively dependent on supplied nitrogen and were not remobilizinglarge internal sources of nitrogen as might occur during bolting andflowering. Plants were grown under conditions where nitrogen wasnon-limiting (i.e., regular fertilization with 10 mM nitrate) and whichreduced the photosynthetic interference of neighboring plants, and thegrowth analysis was terminated when such interference became apparent.All plants analyzed were of the same age, and analysis stated at between0.1 and 0.3 g fresh weight, and continued until the plants wereapproximately six weeks old when the interference of neighboring plantsbecame apparent at the onset of bolting.

6.2.8. Plant Growth Experiment A

Table 2 shows the results of mean total fresh weight determinations forlines Z3-1 and Z3-2 (overexpressing GS1) and for Z17-6 and Z17-7(overexpressing GS3A). These results are expressed graphically in FIG.9, panel A and analyzed statistically in Table 3. All four transgeniclines overexpressing pea cytosolic GS outgrew the control by between 35%and 114%, and this was statistically significant for three lines;Z3-2(P=0.08), Z17-6(P=0.0015) and Z17-7(P=0.013) (Table 3).

                  TABLE 2                                                         ______________________________________                                        Growth Increase of Transgenic Lines                                           Overexpressing Cytosolic GS1 (Z3) or Cytosolic                                GS3a (Z17)                                                                    Experiment A.sup.1                                                                       C       Z3-1    Z3-2  Z17-6  Z17-7                                 ______________________________________                                        Week 4     0.42    0.33    0.42  0.44   0.52                                  Week 5     1.40    1.88    2.36  2.73   1.91                                  Week 6     4.06    5.48    8.67  8.27   6.80                                  % Increase at                                                                            100     135     214   204    150                                   week 6 compared                                                               to control                                                                    ______________________________________                                        Experiment B.sup.1                                                                       C-1*   C-2*   Z3-3  Z3-4 Z17-3 Z17-11                              ______________________________________                                        Week 3     0.12   0.07   0.32  0.24 0.32  0.20                                Week 4     0.60   0.41   1.11  0.77 1.08  1.00                                Week 5     1.19   1.11   1.82  1.36 2.39  1.71                                Week 6     6.49   5.83   9.37  6.04 9.34  9.06                                % Increase at                                                                            100    90     144   93   144   140                                 week 6 compared                                                               to C-1                                                                        ______________________________________                                         .sup.1 Mean total fresh weight (in grams) of transgenic lines and control     measured over a period of three to four weeks immediately prior to the        onset of bolting.                                                             *C1 is control 1 and C2 is control 2.                                    

                  TABLE 3                                                         ______________________________________                                        Growth Increase of Transgenic Lines Over-                                     expressing Cytosolic GS1 (Z3) or Cytosolic                                    GS3A (Z17) with Comparison to Controls By                                     Unpaired T Test                                                               Experiment A                                                                             C       Z3-1    Z3-2   Z17-6 Z17-7                                 ______________________________________                                        week 6     4.06    5.48    8.67   8.27  6.80                                  % Increase at                                                                            100     135     214    204   150                                   week 6 compared                                                               to control                                                                    Number of Plants                                                                         3       6       5      7     6                                     (week 6)                                                                      Standard Error                                                                           0.51    0.75    1.62   0.53  0.53                                  Standard   0.88    1.85    3.62   1.41  1.28                                  Deviation                                                                     "T" for unpaired   1.23(7) 2.10(6)                                                                              4.72(8)                                                                             3.29(7)                               test to control                                                               (df)                                                                          Probability        0.26    0.08   0.0015                                                                              0.013                                 Significance       NS      (*)    **    *                                     ______________________________________                                        Experiment B                                                                            C-1    C-2     Z3-3 Z3-4  Z17-3 Z17-11                              ______________________________________                                        week 6    6.49   5.83    9.37 6.04  9.34  9.06                                % Increase                                                                              100    90      144  93    144   140                                 at week 6                                                                     to C-1                                                                        Number of 7      6       7    7     7     7                                   Plants                                                                        (week 6)                                                                      Standard  0.60   1.07    0.88 0.61  1.06  0.73                                Error                                                                         Standard  1.58   2.61    2.33 1.61  2.81  1.94                                Deviation                                                                     "T" for                  2.70 0.53  2.34  2.72                                unpaired test            (12) (12)  (12)  (12)                                to C-1                                                                        (df)                                                                          Probability              0.019                                                                              0.61  0.038 0.019                               Significance             *    NS    *     *                                   ______________________________________                                         Mean total fresh weight for transgenic lines (in grams) and controls at       week 6. The statistical analysis was done for the final week's measuremen     only, and in the case of experiment II control1 (C1) was selected for the     Ttest. df  degrees of freedom; The probability of the populations being       related was deemed to be highly significant (**) for P(0.001, significant     (*) for P(0.05, and marginally significant ((*)) for P(0.01. NS = not         significant.                                                             

6.2.9. Plant Growth Experiment B

The growth experiment was repeated with different transgenic linescarrying the same GS1 (Z3) and GS3A (Z17) constructions to confirm theresults obtained above, including larger plant populations forstatistical analysis. Table 2 shows the mean data for four time pointsfor transgenic lines Z3-3, Z3-4, Z17-3, and Z17-11, together with twocontrol lines (C1, C2). All lines

except Z3-4 outgrew controls by between 40 and 44% and the difference infresh weights at six weeks was statistically significant (Table 3).These results are also shown graphically in FIG. 9, panel B. It isapparent that the second growth experiment corroborated the results ofthe first, suggesting that ectopic overexpression of wither peacytosolic GS1 or GS3A enhanced growth rate in tobacco; in all linestested GS3A overexpression gave an increase in growth rate which wasstatistically significant increases in growth rate to the transgenictobacco, compared to non-transformed controls.

6.2.10. Qualitative Effect of GS Overexpression on Plant Growth

FIG. 10 demonstrates a qualitative comparison of the growth phenotype ofplants which overexpress GS (Z3-A1 and Z17-B7) to those of controlplants and plants co-suppressed for GS (Z54-A2). The results demonstratethat even low level GS overexpression results in readily discerniblegrowth improvements (FIG. 10, compare the growth of Z17-B7 and Z3-A1with that of control plants). Moreover, these results show that thegrowth improvements are due to GS overexpression and not to the mereengineering of plants with CaMV-35S GS constructs. For example, Z54-A1,which as been engineered with CaMV 35S-GS2 and was co-suppressed for GSexpression, exhibited profoundly poor growth. Furthermore, these resultsdemonstrate that GS activity is a rate limiting step in plant growth asinhibition of this enzyme causes severe phenotypic effects on growth.

6.2.11. Correlation Between GS Activity and Final Fresh Weight and TotalProtein

Experiments were performed to determine whether changes in GS activityassociated with ectopic overexpression or co-suppression of GS genes hadan effect on "final" fresh weight at the end of the vegetative growthphase. Growth analysis was performed on T2 generation plants for a lineco-suppressed by GS2 (Z54-4), a line overexpressing GS1 (Z3-1), a lineoverexpressing GS3A (Zl7-7), and an untransformed tobacco control (SR1).Plants were grown in sand and irrigated periodically with Hoagland'ssolution containing 10 mM KNO₃. At designated time-points, eightindividual T2 plants from each line were weighed and leaf GS activitywas determined for each individual. Analysis of this data reveals alinear relationship between "final" fresh weight and GS specificactivity for all individuals assayed at both 32 days and 43 days (FIG.11A). For example, Z54-4 plants which are co-suppressed for GS activity(27% of wild-type GS activity) weigh one-half as much as controls, whileplants which overexpress GS3A (136% GS activity) or GS1 (284% GSactivity) out-weigh controls by 1.5-times and 2-times, respectively. Forthese same individual T2 plants, a linear relationship also existsbetween total leaf protein (μg protein/gm fresh weight) and leaf GSactivity. Plants expressing the highest levels of GS activity (284%) had1.5-fold higher levels of soluble protein/gram fresh weight compared tocontrols (FIG. 11B). An unpaired T-test analysis of this data revealedthat the GS overexpressing lines (Z3-1,Z17-7) had significantly greaterGS activity, fresh weight, and leaf soluble protein with a p value of<0.0001, with the exception of fresh weight for Z17-7 whose p value was0.0007. Similarly the line co-suppressed by GS2 (Z54-4) hadsignificantly less GS activity, fresh weight, and leaf soluble proteinthan control SR1 with a p value of <0.0001. The GS activity profiles ofthe GS overexpressing T2 lines used in the growth study (Z3,Z17)parallel the parental T0 lines and the T1 progeny, except that the GSactivities were consistently higher in the T2 generation. This is mostlikely due to the fact that some or all of the transgenes becamehomozygous in the T2 generation, as there was no observed segregation ofthe Kan^(R) phenotype associated with the GS transgene. At the end ofthe growth experiment, the transgenic lines overexpressing GS werevisibly greener and dramatically larger than controls.

6.3. Discussion

As genetic engineering begins to assume significance in crop plantimprovement it is becoming increasingly important to understand theparameters critical in the overexpression of selected genes. It isapparent that the overexpression of genes for which there are host planthomologs may be more complex than the overexpression of genes for whichthere are no homologs, such as viral coat protein and BT toxin genes(Powell-Abel et al., 1986, Science 232:738-743; Vaeck et al., 1987,Nature 328:33-37). This is due to the phenomenon of co-suppression inwhich the transgenic plant can detect and silence a transgene to whichthere is a host homolog, perhaps by feedback inhibition or some othermechanism (van der Krol et al., 1990, Plant Cell 2:291-299; Napoli etal., 1990, Plant Cell 2:279-289). Presented here is an effort toectopically overexpress three different pea GS genes for chloroplast orcytosolic GS behind the same constitutive promoter (35S-CaMV) intransgenic tobacco. The effort resulted in overexpression and/orco-suppression that is different for each GS gene. Furthermore, for thetwo genes for cytosolic GS which were successfully overexpressed (GS1and GS3A), the overexpression resulted not only in over production of GSRNA, protein and enzyme, but also in a phenotype of improved nitrogenuse efficiency.

Overexpression of the pea gene for cytosolic GS1 in tobacco gives aclear phenotype of increased GS activity, increased cytosolic GSprotein, and high levels of transgene mRNA. Furthermore, the GS1 proteinassembles into a GS holoenzyme similar in size and charge to native peacytosolic GS. In transgenic plants overexpressing cytosolic GS3A, thesituation is somewhat different. High levels of GS3A transgene mRNA giverise to increased levels of cytosolic GS which are visible on Westernblots. However, the overexpression of GS3A causes the appearance of anovel GS holoenzyme which is larger than the native chloroplastic orcytosolic GS holoenzymes of either pea or tobacco. In these transgenicplants, the cytosolic GS gene was being expressed in a cell type whereit is not normally found (e.g., mesophyll cells), and it was possiblethat the larger GS holoenzyme in the GS3A transgenic leaves was due tothe co-assembly of cytosolic GS subunits with native pre-chloroplastGS2. However, this novel GS3A holoenzyme was shown to be composedexclusively of cytosolic GS subunits and was therefore not due to theco-assembly of transgenic GS3A subunits with endogenous tobaccopre-chloroplastic GS2. Two other possibilities exist. The larger GS3Aholoenzyme may be the result of transgenic GS3A subunits assembling intoa configuration other than their usual octameric structure.Alternatively, the novel GS3A holoenzyme may result from the failure ofthe overexpressed cytosolic subunits to be released from an assemblingchaperonin. Indeed, the close association of GS with groEL-like proteinshas previously been observed in pea (Tsuprun et al., 1992, Biochim.Biophys. Acta 1099:67-73). However, our attempts to dissociate the novelGS3A activity band from a potential chaperonin using ATP wereunsuccessful. Although the novel GS3A holoenzyme must clearly possess GSactivity (from its detection in GS activity gel analysis) it isinteresting to speculate whether or not this novel GS isozyme possessesa similar activity to the native cytosolic GS or chloroplastic GS2holoenzymes on an equimolar basis. If this is the case, it might bepredicted that plants overexpressing 35S-GS3A, and therefore possessingthe novel GS holoenzyme, may have elevated total GS activities. In factthis was not the case; the mean total GS activity (compared towild-type) of four Z17-6 T2 progeny plants (expressing GS3A) was foundto be 100%, and that of four Z17-7 progeny plants was 107% compared towild-type. By contrast, GS activity values obtained for T2 progenies ofZ3-1 and Z3-2 (overexpressing a GS1 native holoenzyme) were 123% and120% respectively, compared to wild-type. This suggests that theassembly in the GS1 subunits in the Z3 overexpressing transformants intoa GS holoenzyme of native size was advantageous to total GS activity.

Here, nitrogen use efficiency was assessed during the vegetative growthstage of transgenic tobacco which successfully overexpressed withercytosolic GS1 or cytosolic GS3A. During vegetative growth there is rapidleaf development characterized by rapid nutrient uptake and themaximization of photosynthetic capacity. Nitrogen is the most frequentlylimiting micronutrient, and the physiology of its uptake and use withinthe plant differs between the vegetative and generative stages. firstlythere is nitrogen incorporation from the soil, its incorporation intoexpanding tissues, and the limitation of losses through photorespirationand subsequently, with the onset of bolting, there is the mobilizationof nitrogen reserves for conversion to seed yield during the generativestage of growth. It is likely that the parameters of nitrogen useefficiency are less complex during the vegetative growth stage ofdevelopment, and our transgenic plant growth analysis has focused onthis stage of growth.

The present findings indicate that ectopically expressed pea cytosolicglutamine synthetase in tobacco provides a considerable advantage in thevegetative growth stage of transgenic tobacco. Plants which overexpresseither cytosolic GS1 or GS3A ectopically (i.e., in all cell types) yielda higher total fresh weight that controls. It was particularly strikingthat all GS3A expressing lines (Z17) had higher total fresh weights thancontrols at six weeks and these were always statistically significant.In each case there was a less than a 5% chance that the differencebetween control and transgenic lines was due to sample variance. For theGS1 expressing lines analyzed (Z3), three out of four outgrew controlsand for two of these the difference was statistically significant at the10% level. This increased use efficiency of nitrogen may enable crops tobe similarly engineered to allow better growth on normal amounts ofnitrogen or cultivation with lower fertilizer input, or alternatively onsoils of poorer quality and would therefore have significant economicimpact in both developed and developing agricultural systems.

Although GS-overexpression has previously been attempted in transgenictobacco (Eckes et al., 1989, Mol. Gen. Genet. 217:263-268; Hemon et al.,1990, Plant Mol. Biol. 15:895-904; Hirel et al., 1992, Plant Mol. Biol.20:207-218; Temple et al., 1993, Mol. Gen. Genet. 236:315-325), this isthe first report in which overexpression of GS is correlated with asignificant increase in GS activity and an improvement in plant growthand nutritional characteristics. Temple et al. reported increases in GSmRNA and protein, but no corresponding increase in GS activity in thetransgenic plants (Temple et al., ibid). Hemon et al. reported increasedlevels of GS mRNA in transgenic plants engineered with GS expressionconstructs, but found no corresponding increase in GS protein or enzymeactivity (Hemon et al., ibid). In two other reports, overexpression ofGS genes in transgenic plants did result in increased levels of GSenzyme, but the studies reported no phenotypic effects of GSoverproduction (Eckes et al., ibid; Hirel et al., ibid). There is onereport of overexpression of an alfalfa GS gene improving plant growthrate by about 20% (Eckes et al., 1988, Australia Patent Application No.AU-A-17321/88). However, this reported improvement appears to be limitedto growth under low-nitrogen conditions only. Identically engineeredplants were reported to show no phenotypic changes, as compared tocontrol plants, in a subsequent analysis carried out on a nitrogennon-limiting medium (Eckes et al., 1989, Mol. Gen. Genet. 217:263-268).In addition, there is no report that the faster rate of growth resultsin difference in final fresh weight between the engineered and controlplants. In contrast to these earlier studies, the instant inventiondemonstrate unequivovally that, regardless of the nitrogen conditions,GS overexpression can improve growth, yield, and/or nutritionalcharacterisitics of plants.

The agricultural utility of the instant invention is directly relevantto crop species in which the vegetative organs are harvested, and theseinclude all forage crops, potato, sugar beet, and sugar cane as well astobacco. Within a week of the final fresh weight recordings presentedhere, plants started to undergo internode extension, and the standarddeviation of subsequent fresh weight measurements for each populationincreased as a result of the differing physiological stage of plants.Whether the increased vegetative growth rate would also lead to asignificant seed yield advantage is an important question which remainsto be answered. The physiological parameters relevant to seed yield andseed nitrogen content include not only the efficiency of nitrogenuptake, but also the remobilization of reserves at the onset of bolting,and the consequences of field population density. Such studies would bebetter undertaken in a transgenic species which has been selected forseed yield and for which there is some understanding of yieldphysiology.

The finding that co-suppression of endogenous tobacco GS by genesencoding chloroplastic GS2 and cytosolic GS3A of pea, but not bycytosolic GS1 is also intriguing. This is especially so as pea GS2suppresses only the tobacco chloroplastic GS2 form while GS3A suppressesboth tobacco chloroplastic GS2 and cytosolic GS. Previously, Petuniachalcone synthase and dihydroflavanol-4-reductase have been shown toco-suppress both endogenous and transgenes in transgenic Petunia (vander Krol et al., 1990, Plant Cell 2:291-299; Napoli et al., 1990, PlantCell 2:279-289). More recently it has been reported that either the 5'or the 3' end of the chalcone synthase gene was sufficient to causeco-suppression, but that a promoter-less gene was not (Jorgensen, 1992,Agbiotech News and Information Sept:1992), suggesting the necessity oftranscriptional initiation. Transient ectopic sequence pairing has beeninvoked as a possible mechanism for gene silencing and this may dependon the unwinding of DNA presumably associated with the initiation oftranscription (Jorgensen, 1990, Trends in Biotechnology 8:340-344;Jorgensen, 1991, Trends in Biotechnology 9:255-267; Jorgensen, 1992,Agbiotech News and Information Sept:1992). From the present findings onpea GS gene expression it appears that the co-suppression phenomenondoes not depend on perfect sequence homology at the nucleotide level.

Increasing nitrogen use by modifying the expression of nitrogenassimilatory enzymes may also be a feasible approach to enhancing yieldsin transgenic crop plants such as corn. The efficiency of nitrogen usein crops is measured as enhanced yields and is therefore an agriculturalmeasure. This kind of adaptation or specialization would be of no realadvantage to wild type plants which depend for their survival on adiversity of responses to environmental conditions and not on higheryields (Sechley et al., 1992, Int. Rev. Cyt. 134:85-163). Therefore,increases in crop yield may be more easily realized through geneticengineering methods such as those described herein, rather than byconventional breeding methods.

7.0. EXAMPLE

Ectopic Overexpression of Asparagine Synthetase in Plants Causes anIncrease in Plant Growth Phenotype

The following study concerns the manipulation of AS gene expression inplants with the aim of increasing asparagine production and testing theeffects on plant growth. There are several features of asparagine whichmake it preferable to glutamine as a nitrogen transport/storage compoundand hence the increased assimilation of nitrogen into asparagine may bevaluable in vivo. Asparagine is a long-distance nitrogen transportcompound with a higher N:C ratio than glutamine. It is therefore a moreeconomical compound for nitrogen transport. In addition, asparagine ismore stable than glutamine and can accumulate to high levels in vacuoles(Sieciechowicz et al., 1988, Phytochemistry 27:663-671; Lea and Fowden,1975, Proc. R. Soc. Lond. 192:13-26). In developing pea leaves,asparagine is actively metabolized, but in mature leaves that no longerneed nitrogen for growth, asparagine is not readily metabolized and isre-exported (in the phloem) from the leaf to regions of active growthsuch as developing leaves and seeds (Sieciechowicz et al., 1988,Phytochemistry 27:663-671; Ta et al., 1984, Plant Physiol 3574:822-826). AS is normally only expressed in the dark (Tsai andCoruzzi, 1990, EMBO J. 9:323-332) therefore 35-AS1 is expressedconstitutively and not only ectopically expressed in regard to celltype, but also in regard to temporal expression. Thus, the studiespresented here examined whether the ectopic overexpression of AS in allcell-types in a light-independent fashion would increase asparagineproduction. Also tested here was whether the increased asparagineproduction provides an advantage in the nitrogen use efficiency andgrowth phenotype of transgenic plants.

In addition to overexpression wild-type AS, the present study alsoexamined the ectopic overexpression of a modified form of the AS enzyme(glnΔAS1) which was missing the glutamine-binding domain. A questionaddressed by this study was whether ectopic overexpression of a glnΔAS1form of the enzyme might produce a novel plant AS enzyme with enhancedammonia-dependent AS activity or whether such a mutation may have adominant-negative effect (Herskowitz, 1987, Nature 329:219-222) due toco-assembly with endogenous wild-type AS subunits to form a heterodimer(Rognes, 1975, Phytochemistry 14:1975-1982; Hongo and Sato, 1983,Biochim et Biophys Acta 742:484-489). The analysis of the transgenicplants which ectopically express pea AS, demonstrated an increasedaccumulation of asparagine and an improved growth phenotype (in the caseof 35S-AS1), and an increased accumulation of asparagine but accompaniedby a detrimental effect on growth phenotype (in the case of35S-glnΔAS1). These results indicate that it is possible to manipulatenitrogen metabolism and growth phenotype by ectopic overexpression of ASgenes. Because nitrogen is often the rate-limiting element in plantgrowth and typically applied to crops several times during the growingseason, designing molecular technologies which improve nitrogen useefficiency in crop plants is of considerable interest to agriculture.

7.1. Materials and Methods 7.1.1. AS Gene Constructs

The AS1 cDNA previously cloned from pea (Tsai and Coruzzi, 1990, EMBO J9:323-332) was transferred from pTZ18U to the EcoRl site of pBluescriptKS- (Stratagene). A glnΔAS1 deletion mutant was constructed using"inside-outside" PCR (Innis et al., 1990, PCR Protocols: A guide oMethods and Applications. New York, Academic Press p.1-461). Codingsequence corresponding to amino acids 2-4 (CGI) was deleted from theamino terminus of the AS1 cDNA, leaving the initiating methionine andthe untranslated leader intact. This deletion corresponded to thepresumed glutamine-binding domain of the AS enzyme comprising aminoacids MCGI which have been defined for animal AS (Pfeiffer et al., 1986,J. Biol. Chem. 261:1914-1919; Pfeiffer et al., 1987, J. Biol. Chem.252:11565-11570). cDNAs corresponding to wild-type AS1 and glnΔAS1 werethen transferred from pBluescript to the binary expression vector pTEV5.This vector contains the CaMV 35S promoter (from -941 to +26), amultiple cloning site, and the nopaline synthase terminator. FIG. 12shows details of the binary vector constructions containing the AS1cDNAs pZ127 (NRRL Accession No. B-21335) and glnΔAS1 cDNA pZ167 (NRRLAccession No. B-21336), which were transformed into tobacco.

7.1.2. Plant Transformations

Binary vector constructions were transferred into the disarmedAgrobacterium strain LBA4404 and subsequently into Nicotiana tabacum SR1using standard procedures described elsewhere (Bevan, 1984, NucleicAcids Res. 12:8711-8721; Horsch et al., 1985, Science 227:1229-1231).

7.1.3. RNA Analysis of Transformants

RNA was isolated using "RNA matrix" from Bio101 and total RNA waselectrophoresed as previously described (Thomas, 1983, Methods Enzymol.100:255-266). Gels were capillary blotted onto Hybond-N nylon membrane(Amersham). cDNAs were labeled using the random primer plus extensionreagent labeling system supplied by NEN. Hybridizations were done inaqueous solution and blots were washed in 0.1× SSPE, 0.1% SDS. Northernblots were probed with the pea AS1 cDNA, pAS1 (Tsai and Coruzzi, 1990,EMBO J 9:323-332).

7.1.4. Extraction of Free Amino Acids

Tobacco leaf tissue samples were frozen in liquid nitrogen and extractedin 10 mls of extraction media consisting of methanol:chloroform:water(12:5:3, v/v/v). The homogenate was centrifuged at 12,000×G for 15minutes. The pellet was extracted again and the supernatants werecombined. Addition of 2.5 ml chloroform and 3.8 ml of distilled water tothe supernatant induced separation. The methanol:water phase wascollected and dried under vacuum and redissolved in 1 ml of distilledwater. The solution was filtered by passing it through a 0.45 μm nylonfilter microcentrifuge tube filter system centrifuged at 12,000 g for 2min.

7.1.5. HPLC Determination of Amino Acid Pools

The amino acids were determined as o-phthaldialdehyde (OPA) derivativeson a Microsorb Type O AA Analysis column (Rainin) using a DuPont HPLCinstrument. Sample (100 μL) was derivatized with 100 μl of OPA workingreagent. After 2 min of derivatization, 50 μL of the derivatized samplewas injected. This gradient was produced using two eluents: A. 95% 0.1 Msodium acetate (pH 7.2) with 4.5% methanol and 0.5% tetrahydrofluoran;B. 100% methanol. Eluents were filtered and degassed with He before use.Detection of OPA derivatized amino acids was accomplished with a UVspectrophotometer at 340 nm. Each determination was done twice and thevalues represent the average.

7.1.6. Plant Growth Conditions

Progenies of primary transformants characterized as expressing highlevels of either as AS1 mRNA or the mutated glnΔAS1 mRNA were germinatedon MS-medium containing 100 μg/ml kanamycin. After 14 days, kanamycinresistant seedlings were transferred to 4 inch pots filled with whitesand, which were covered with saran wrap for approximately one week toprevent excessive transpiration and enable seedlings to becomeestablished. Pots were irrigated periodically with 1X Hoagland'ssolution containing 10 mM potassium nitrate as the only nitrogen source.Subsequently, between three and seven plants were sacrificed for freshweight determination each week, continuing for a period of four weeksuntil shading of neighbors was apparent. Plants were grown under alight-dark cycle of 16-8 h with a temperature cycle of 24-18° C. Daytimelight intensity was 1000 lux.

7.2. Results 7.2.1. Construction of Transgenic Plants Expressing pea AS1and glnΔAS1

The pea AS1 cDNA (Tsai and Coruzzi, 1990, EMBO J 9:323-332) expressedfrom the 35S-CaMV promoter was transferred into transgenic tobacco (SeeFIG. 12 and Section 7.1 Material and Methods) and five independentprimary transformants (Z127; 1-5) were shown to express high levels ofthe AS1 mRNA (see below). Three independent transgenic lines (Z167;1-3)which contained the AS1 cDNA with a deletion in the glutamine bindingdomain (glnΔAS1) were also shown to contain high levels of transgene RNA(see infra).

7.2.2. Northern Analysis of Transformants Expressing AS1 and glnΔAS1

Northern blot analysis of RNA extracted from transgenic plants wereundertaken to identify plants in which the 35S-AS1 transgene wasexpressed at high levels (FIG. 13). As a positive control, RNA for ASwas detected in leaves of pea plants grown in the dark (FIG. 13, lanePL). By contrast, no AS mRNA was detected in leaves of light-grownwild-type tobacco (FIG. 13, TL). Previous studies have shown thattobacco AS mRNA is expressed exclusively in tissues of plants grown thedark (Tsai and Coruzzi, 1991, Mol Cell Biol 11:4966-4972). Thetransformants which overexpress AS1 (Z127-1, -3, -4, and -5) allcontained high levels of AS1 mRNA, even though these plants were grownin the light (FIG. 13). Thus, the 35S CaMV promoter producesconstitutive expression of pea AS1, whereas the endogenous AS mRNA isnot expressed in tobacco leaves in the light. The glnΔAS1 transformantsalso showed constitutive high level expression of mRNA (Z167-2, -3, and-4), compared to tobacco controls (FIG. 13). Because the AS enzyme isnotoriously unstable, the AS enzyme has never been purified tohomogeneity and antibodies for plant AS were not available for ASprotein analysis. In addition, in vitro assay detected no AS activitydue to enzyme instability.

7.2.3. Amino Acid Analysis of Transgenic lines expressing AS1 andglnΔAS1

Based on the Northern results, two independent transgenic lines whichshowed high levels of AS1 mRNA (Z127-1 and Z127-4) were selected forfurther analysis. Similarly, lines Z167-2 and Z167-4 were selected ashigh-expressing representatives of the glnΔAS1 construction. The T2progenies of these plants were subjected to amino acid and growthanalysis described below.

7.2.4. AS1-Overexpressing Lines

Both Z127 lines selected (Z127-1 and Z127-4) showed increased levels ofasparagine (10- to 100-fold higher than wild-type controls) (Table 4).The variation apparent among the individual T2 plants most likelyreflects the homozygosity or heterozygosity of individuals, and theapproximate 2:1 ratio of intermediate:high asparagine levels wouldsubstantiate this assertion. In all cases, however, a considerableincrease in asparagine is seen extending up to nearly 100-times thecontrol concentration. Interestingly, there is a corresponding reductionin glutamine concentrations in these plants (although the Z127-4 data isskewed by a single high value) and this reflects the use of glutamine asa substrate in the AS reaction; equally predictable is the reduction inconcentration of the other substrate aspartate. Somewhat unexpected,however, is the reduced concentration of glutamate in the Z127 lines.From biochemical predictions and from the data collected for the otherthree amino acids involved in the AS reaction, an increase in glutamatewould have been predicted. The apparent reduction in glutamate may bethe result of its high turnover rate due to its use as a substrate inseveral related processes such as transamination.

7.2.5. glnΔAS1-Overexpressing Lines

In the two lines selected which overexpress glnΔAS1, the questionassessed was whether the deletion of the glutamine-binding domain of ASwould have a dominant-negative effect on asparagine biosynthesis. Thedata collected for these lines (Z167-2 and Z167-4) is somewhat difficultto interpret due to the variation of data values (Table 4). However, inalmost every case there is a substantial increase in asparagineconcentration, ranging from 3- to 19-fold compared to wild-typenon-transgenic tobacco. These results suggest that the transgenic lineshave the ability to accumulate asparagine with little effect onaspartate, glutamate or glutamine pools. One possibility is that theglnΔAS1 enzyme is able to synthesize asparagine directly from ammoniaand aspartate.

7.2.6. Plant Growth Experiment on Transformants Expressing AS1 andglnΔAS1

Growth analysis was undertaken using individual transgenic T2 plantsgrown in white sand. These studies were aimed at assessing growth rateunder conditions which minimized interference from neighboring plants.For this reason, fresh weight measurements were taken only during thevegetative stage of growth (up to six weeks post germination). Duringthis period, plants undergo rapid growth and are exclusively dependenton supplied nitrogen and do not remobilize internal nitrogen sources asmight occur during bolting and flowering. Plants were grown underconditions where nitrogen was non-limiting (i.e., regular fertilizationwith 10 mM nitrate) and which reduced the photosynthetic interference ofneighboring plants. The growth analysis was terminated when suchinterference became apparent. All plants analyzed were of the same ageat each time point, and analysis started at between 0.1 and 0.3 g freshweight/plant, and continued until the plants were approximately sixweeks old when the interference of neighboring plants became apparentand bolting was imminent.

                  TABLE 4                                                         ______________________________________                                        Amino Acid Analysis in Transgenic Lines                                       Overexpressing AS1 or glnΔAS1                                           PLANT ID     ASN    GLU        GLN  ASP                                       ______________________________________                                        CONTROL                                                                       C            34     1399       309  1935                                      C            38     1425       405  1861                                      C            36     965        425  2015                                      C            47     1526       275  1720                                      mean         39     1335       353  1883                                      AS1 wild-type                                                                 Z127-1-A     553    228        14   182                                       Z127-1-B     3399   808        60   922                                       Z127-1-C     213    525        81   240                                       Z127-1-D     487    537        17   264                                       Z127-1-E     3159   983        43   796                                       mean         1562   616        43   481                                       Z127-4-A     1105   838        132  451                                       Z127-4-B     902    2947       389  1092                                      Z127-4-C     373    1606       17   678                                       Z127-4-D     4109   691        923  1664                                      mean         1622   1520       365  971                                       glnΔAS1                                                                 Z167-2-A     684    838        352  761                                       Z167-2-B     1341   2947       944  3119                                      Z167-2-C     173    1606       1224 1946                                      mean         733    1797       840  1942                                      Z167-4-A     47     691        75   948                                       Z167-4-B     109    864        346  1491                                      Z167-4-C     137    1313       714  1705                                      Z167-4-D     165    1534       838  2069                                      mean         114    1100       493  1553                                      ______________________________________                                         Amino acid concentrations are in nmol/gram fresh weight                  

                  TABLE 5                                                         ______________________________________                                        Growth Increase of Transgenic Lines                                           Overexpressing AS1 or glnΔAS1                                                    C-1  C-2    Z127-1  Z127-4                                                                              Z167-2                                                                              Z167-4                               ______________________________________                                        3          0.12   0.07   0.28  0.12  0.11  0.19                               4          0.60   0.41   1.30  0.51  0.31  0.57                               5          1.19   1.11   1.87  1.72  0.71  0.99                               6          6.49   5.83   8.63  7.16  3.83  6.13                               % increase at                                                                            100    90     133   110   59    94                                 week 6 compared                                                               to C-1                                                                        ______________________________________                                         Total fresh weight means (in grams) of transgenic lines and controls          measured over a period of three to four weeks immediately prior to the        onset of bolting.                                                        

Tables 5 and 6 show the results of mean total fresh weightdeterminations for lines Z127-1 and Z127-4 (overexpressing wild-typeAS1) and Z167-2 and Z167-4 (overexpressing glnΔAS1), and these resultsare expressed graphically in FIG. 13. Transgenic lines overexpressingwild-type AS grew 133% and 110% compared to control (100%) (Table 5),although in neither case was this statistically significant whenanalyzed by unpaired T-test (Table 6). Transgenic lines overexpressingthe glnΔAS1 construction (Z167) did not perform comparably. The Z167-4plants which survived until the sixth week were indistinguishable ingrowth from controls, and the Z167-2 plants which survived, were muchsmaller than controls (P--0.041; significant at the 5% level) (Tables 5and 6, and see also FIG. 14). Comparing the three different lines in theexperiment, it was of interest that a greater proportion of kanamycinresistant Z167 plants died. Typically the Z167 plants were slow togerminate and looked unhealthy when grown in pots. This was clearlyreflected in the fresh weight data collected for Z167-2, although lessapparent for the Z167-4 data, suggesting that the glnΔAS1 gene productdid indeed have a dominant-negative effect on plant growth.

                  TABLE 6                                                         ______________________________________                                        Growth Increase of Transgenic Lines                                           Overexpressing AS1 or glnΔAS1 with Comparison                           to Controls By Unpaired T Test                                                         C-1  C-2    Z127-1  Z127-4                                                                              Z167-2                                                                              Z167-4                               ______________________________________                                        Week 6     6.49   5.83   8.63  7.16  3.83  6.13                               % Increase at                                                                            100    90     133   110   59    94                                 week 6 compared                                                               to control 1                                                                  Number of  7      6      7     7     3     5                                  Plants                                                                        (week 6)                                                                      Standard   0.60   1.07   1.15  0.88  0.92  0.85                               Error                                                                         Standard   1.58   2.61   3.05  2.34  1.60  1.89                               Deviation                                                                     "T" for                  1.65  0.63  2.43  0.35                               unpaired test            (12)  (12)  (8)   (10)                               to control-1 (df)                                                             Probability              0.125 0.54  0.041 0.731                              Significance             NS    NS    *     NS                                 ______________________________________                                         Total fresh weight means for transgenic lines (in grams) and controls at      week 6. The statistical analysis was done for the final week's measuremen     only and control1 was selected for the Ttest df  degrees of freedom; The      probability of the populations being related was deemed to be significant     (*) for P < 0.05; NS  not significant                                    

7.3. Discussion

Reported here are studies in which AS is ectopically overexpressed intransgenic plants to test the effects of this manipulation on primarynitrogen assimilation and on plant growth. In particular, thecell-specific expression pattern of AS were altered and the regulationof AS with regard to light was also modified. In wild-type plants, AS isnormally only expressed in the phloem (Tsai, 1991, Molecular BiologyStudies of the Light-Repressed and Organ-Specific Expression of PlantAsparagine Synthetase Genes. Ph.D. Thesis, The Rockefeller University,New York, N.Y.), and its expression is dramatically repressed by lightin both photosynthetic and non-photosynthetic tissues (Tsai and Coruzzi,1990, EMBO J 9:323-332; Tsai and Coruzzi, 1991, Mol Cell Biol11:4966-4972). Here, the wild-type AS1 of pea and a mutated form of AS1(glnΔAS1) were expressed under the control of a constitutive promoter(35S-CaMV) in transgenic tobacco so that AS1 is expressed in all celltypes, in a light-independent fashion. The physiological significance ofconstitutively expressing AS1 in cells where it is not normallyexpressed may have considerable impact on plant nitrogen metabolism. Forexample, asparagine is involved in photorespiratory nitrogen recycling(Givan et al., 1988, TIBS 13:433-437; Ta et al., 1984, Plant Physiol74:822-826), thus the ectopic expression of AS in photosynthetic cellsmay have dramatic impact on photorespiration. Furthermore, theexpression of an ammonia dependent AS enzyme in this context may aid inthe reassimilation of photorespiratory ammonia.

Four independent transgenic tobacco lines expressing 35S-AS1 have beenshown to express a wild-type pea AS1 transgene constitutively. Two lineswere analyzed further (Z127-1 and Z127-4) and it was shown that theexpressed AS1 gene was functional since free asparagine accumulated tohigh levels in transgenic leaf tissue; typically transgenic lines Z127-1and Z127-4 accumulated between 10- and 100-fold more asparagine thancontrol untransformed tobacco lines. These increased asparagine levelswere predictably accompanied by a reduction in the AS substrates,glutamine and aspartate. However, it may still be possible to channelmore inorganic nitrogen into the nitrogen transport compound asparagineby providing higher endogenous levels of glutamine, a substrate for AS.

The plant growth experiment on the Z127 transgenic plants was aimed atdetermining whether the accumulation of asparagine in the AS1overexpressing plants might have a positive effect on growth during thevegetative stage of plant development. The rapid leaf development whichoccurs during vegetative growth imposes a strong demand for nutrientavailability and nitrogen is typically the most critical nutrient atthis time due to the synthesis of new proteins in expanding andenlarging tissues. Nitrogen assimilated and accumulated at this time issubsequently recycled in the plant and deposited in seed reserves; aswell as being a major long-distance transport amino acid, asparaginealso plays an important role in the formation of seed reserves (Dilworthand Dure, 1978, Plant Physiol 61:698-702; Sieciechowicz et al., 1988,Phytochemistry 27:663-671). The two Z127 lines were found to outgrowuntransformed controls over a six week period up to the end ofvegetative growth and conferred a 10% and a 33% growth advantage.However, these figures were not statistically significant when a T-testis performed. Thus, although the plants make 10- to 100- fold higherlevels of asparagine, it is possible that glutamine levels are limitingrelative to increases in growth. Also presented here is the finding thatoverexpressing GS in transgenic tobacco can confer a greater growthadvantage which is statistically significant (supra). As glutamine is asubstrate for asparagine biosynthesis both are pivotal amino acids inthe primary assimilation of inorganic nitrogen. It can therefore beanticipated that creating transgenic lines which express both GS and ASat high levels (by crossing AS and GS overexpressers) may have even moreadvantageous growth traits than either parent. In particular, theapproaches disclosed here have the advantage that assimilation intransgenic lines will not be restricted to a few cell types, enablingavailable nitrogen in all plant cells to be utilized. The ectopicoverexpression of both GS and AS in a single plant may have theadvantage of avoiding glutamine accumulation; since glutamine is anactive metabolite in the presence of high concentrations of glutaminemay upset cell metabolism. By contrast, asparagine is a relatively inertcompound able to store nitrogen economically. In addition, asparagine isformed in a reaction which liberates a molecule of glutamate thenavailable to accept a further unit of ammonia (Lea and Fowden, 1975,Proc. R. Soc. Lond. 192:13-26).

In addition to the ectopic overexpression of wild-type AS, the plantglutamine-dependent AS was modified in an attempt to enhance itsammonia-dependent activity. In particular, it has been shown in animalsthat antibodies to the glutamine-binding domain of AS inhibitglutamine-dependent AS activity present on the same AS polypeptide, yetenhance the ammonia-dependent activity (Pfeiffer et al., 1986, J. Biol.Chem. 261:1914-1919; Pfeiffer et al., 1987, J. Biol. Chem.252:11565-11570). By analogy, a site-specific mutant was created in apea AS1 cDNA (Tsai and Coruzzi, 1990, EMBO J 9:323-332) which mutationspecifically deleted the three amino acids required for glutaminebinding (glnΔAS1). By introducing this glnΔAS1 into transgenic plants,it might be possible to enhance the ammonia-dependent AS activity and/orinhibit the endogenous glutamine-dependent AS activity through subunitpoisoning and the formation of heterodimers of wild-type and mutantsubunits. Two independent transgenic lines, Z167-2 and Z167-4, whichoverexpress the glnΔAS1 transgene were found to be capable ofaccumulating asparagine levels approximately 3- to 19-times greater thanuntransformed tobacco controls. The activity of the glnΔAS1 gene inassimilating asparagine is suggestive of the modified enzyme having thecapability of utilizing a nitrogen substrate other than glutamine (e.g.,ammonia). By analogy to the known ammonia-dependent AS activities of theE. coli AsnA gene and mammalian AS, the high levels of asparagine in thetransgenic plants which express the mutated plant glnΔAS1 enzyme suggestthat the glnΔAS1 enzyme can assimilate ammonia directly into asparagineand therefore bypass GS in primary nitrogen assimilation. If thissuggestion is correct, it is also apparent that the glnΔAS1 gene is notas efficient in synthesizing asparagine as the overexpressed wild-typeAS1, based on the relative levels of asparagine in these transgenicplants (Z167 vs. Z127).

Transgenic lines expressing glnΔAS1 (Z167-2 and Z167-4) did not outgrowuntransformed controls; indeed they typically grew more poorly thanuntransformed plants as evidenced by the performance of Z167-2 and thehigher proportion of Z167 plants to die before the end of theexperiment. It is curious that these plants should accumulate 3- to19-fold higher levels of asparagine in their leaves, yet grow morepoorly. Plant AS is believed to assemble as a homodimer (Rognes, 1975,Phytochemistry, 14:1975-1982). In leaf mesophyll tissue where wild-typeAS is not normally expressed, the glnΔAS1 form is able to self-assembleinto homodimers and form an enzyme capable of generating asparagine. Inphloem cells, however, glnΔAS1 subunits may co-assemble with wild-typeAS subunits, thereby inactivating wild-type AS as a dominant-negativemutation (Herkowitz, 1987, Nature 329:219-222). In the glnΔAS1 plants,asparagine synthesized in leaf mesophyll cells may be unable to betransported to and loaded into the phloem and this could account for thepoor growth phenotype of these transgenic lines. These observationshighlight the specialization of cell-type function, and cell-specificgene expression of nitrogen metabolic genes and their impact on plantnitrogen metabolism.

8. DEPOSIT OF MICROORGANISM

The following microorganism are deposited with the Agricultural ResearchCulture Collection, Northern Regional Research Center (NRRL), Peoria,Ill. and are assigned the following accession numbers:

    ______________________________________                                        Strain          Plasmid  NRRL Accession No.                                   ______________________________________                                        Escherichia coli, Z3                                                                          pZ3      B-21330                                              Escherichia coli, Z9                                                                          pZ9      B-21331                                              Escherichia coli, Z17                                                                         pZ17     B-21332                                              Escherichia coli, Z41                                                                         pZ41     B-21333                                              Escherichia coli, Z54                                                                         pZ54     B-21334                                              Escherichia coli, Z127                                                                        pZ127    B-21335                                              Escherichia coli, Z167                                                                        pZ167    B-21336                                              ______________________________________                                    

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

Various publication are cited herein, the disclosure of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of producing a transgenic plant havingan improved agronomic or nutritional characteristic, which methodcomprises:I) identifying a transgenic plant overexpressing a nitrogenassimilation/metabolism enzyme from among transgenic plants having agene construct comprising a gene encoding a nitrogenassimilation/metabolism enzyme operably linked to a plant promoter sothat the nitrogen assimilation/metabolism enzyme is ectopicallyoverexpressed in transgenic plants, II) screening the transgenic plantoverexpressing the nitrogen assimilation/metabolism enzyme for animproved agronomic or nutritional characteristic under nitrogennon-limiting growth conditions, and III) selecting the transgenic planthaving an improved agronomic or nutritional characteristic;wherein thenitrogen assimilation/metabolism enzyme is cytosolic glutaminesynthetase, and the improved agronomic or nutritional characteristic ofthe transgenic plant is a: i) faster rate of growth, ii) greater freshor dry weight at maturation, iii) greater fruit or seed yield, iv)greater total plant nitrogen content, v) greater fruit or seed nitrogencontent, vi) greater free amino acid content in the whole plant, vii)greater free amino acid content in the fruit or seed, viii) greaterprotein content in seed or fruit, or ix) greater protein content in avegetative tissue,than that of a progenitor plant which does not containthe gene construct, when the transgenic plant having the improvedagronomic or nutritional characteristic and the progenitor plant arecultivated under identical nitrogen non-limiting growth conditions. 2.The method of claim 1, wherein the plant promoter is a strong,constitutively expressed plant promoter.
 3. The method of claim 2,wherein the plant promoter is CaMV 35S promoter.
 4. The method of claim2, wherein the nitrogen assimilation/metabolism enzyme is root-specificglutamine synthetase.
 5. The method of claim 1, wherein the geneconstruct is the 35S-GS gene construct of pZ3, pZ9, or pZ17.
 6. Atransgenic plant having a gene construct comprising a gene encoding anitrogen assimilation/metabolism enzyme operably linked to a plantpromoter so that the nitrogen assimilation/metabolism enzyme isectopically overexpressed in the transgenic plant, and the transgenicplant exhibits:i) faster rate of growth, ii) greater fresh or dry weightat maturation, ii) greater fresh or dry weight at maturation, iii)greater fruit or seed yield, iv) greater total plant nitrogen content,v) greater fruit or seed nitrogen content, vi) greater free amino acidcontent in the whole plant, vii) greater free amino acid content in thefruit or seed, viii) greater protein content in seed or fruit, or ix)greater protein content in a vegetative tissue,than a progenitor plantwhich does not contain the gene construct, when the transgenic plant andthe progenitor plant are cultivated under identical nitrogennon-limiting growth conditions, wherein the nitrogenassimilation/metabolism enzyme is cytosolic glutamine synthetase.
 7. Thetransgenic plant of claim 6, the plant promoter is a strong,constitutively expressed plant promoter.
 8. The transgenic plant ofclaim 7, wherein the plant promoter is CaMV 35S promoter.
 9. Thetransgenic plant of claim 8, wherein the gene construct is the 35S-GSgene construct of pZ3, pZ9, or pZ17.
 10. The transgenic plant of claim7, wherein the nitrogen assimilation/metabolism enzyme is root-specificglutamine synthetase.
 11. A seed of the transgenic plant of any one ofclaims 6-9 or 10, wherein the seed has the gene construct.
 12. Aprogeny, clone, cell line or cell of the transgenic plant of any oneclaims 6-9 or 10 wherein said progeny, clone, cell line or cell has thegene construct.